Basb027 proteins and genes from moraxella catarrhalis, antigens, antibodies, and uses

专利摘要:
The present invention relates to BASB027 polypeptides and polynucleotides encoding BASB027 polypeptides and to methods for producing such polypeptides by recombinant techniques. The invention also relates to the diagnostic, prophylactic and therapeutic uses of the compounds of the invention.
公开号:KR20010052552A
申请号:KR1020007013705
申请日:1999-05-31
公开日:2001-06-25
发明作者:까를로따 비날스 와이 데 바솔즈
申请人:장 스테판느；스미스클라인 비이참 바이오로지칼즈 에스.에이.；
IPC主号:

专利说明:

JASS027 protein and genes, antigens, antibodies, and uses thereof from Moraxella catalis {BASB027 PROTEINS AND GENES FROM MORAXELLA CATARRHALIS, ANTIGENS, ANTIBODIES, AND USES}
Moraxella catalis (also called Branhamella catarrhalis) is a Gram-negative bacterium that is often isolated from the human upper bronchus. These bacteria have several etiologies, the main ones being otitis media in infants and children, and pneumonia in older people. These bacteria are also responsible for sinusitis, pathogens and less frequently occurring invasive diseases.
Otitis media is a serious child disease, considering both its incidence and possible sequelae. In the United States, more than 3.5 million cases are reported each year, and 80% of children are estimated to have at least one symptom of otitis media before they reach age 3. Klein, JO (1994), Clin.Inf.Dis 19 : 823]. If left untreated or chronic, these disorders can cause hearing loss either temporarily (when fluid builds up in the middle ear) or permanently (when the auditory nerve is damaged). In infants, this loss of hearing can cause them to learn to speak late.
Three bacterial species are primarily isolated from the middle ear of a child with otitis media: Streptococcus pneumoniae, non typeable Haemophilus influenzae (NTHi), and Moraxella catalys. They are present at 60 to 90% in the case of otitis media. A review of recent studies shows that in the case of otitis media, both Streptococcus pneumoniae and NTHi are present in about 30% and Moraxella catalis in about 15%. , TF (1996) Microbiol. Rev. 60: 267]. Other bacteria (Hemophilus influenza type B, S. pyogenes, etc.) can be isolated from the middle ear, but the frequency is very low (about 2% or less during the onset).
Epidemiological data shows that for pathogens found in the middle ear, colonization of the upper bronchus is an absolute prerequisite for the development of otitis media; Others indicate that disease is required to induce [Dickinson, DP et al. (1988) J. Infect. Dis. 158: 205, Faden, HL et al. (1991) Ann. Otorhinol. Laryngol. 100: 612. These are important for initiating the inflammatory process after allowing the bacteria to move through the eustachian tube to the middle ear. These factors are currently unknown. It is hypothesized that transient modifications of the immune system after viral infection may render control of bronchial colonization, for example, Faden, HL et al (1994) J. Infect. Dis. 169: 1312. Another explanation is that exposure to surrounding factors enables more influential colonization of some children, who subsequently become susceptible to the development of otitis media due to the continued presence of middle ear pathogens. , TF (1996) Microbiol. Rev. 60: 267].
The immune response to Moraxella catalis is rarely characterized. Analysis of strains isolated from the nasopharynx of babies of age 0-2 years old indicates that these babies frequently receive and remove new strains. These results indicate that an effective immune response against the bacterium is exhibited by colonized children. Faden, HL et al (1994) J. Infect. Dis. 169: 1312.
In most adults tested, bactericidal antibodies have been identified. See Chapman, AJ et al. (1985) J. Infect. Dis. 151: 878. Strains of Moraxella catalis show diversity in their ability to inhibit serum bactericidal activity; In general, strains isolated from patients suffering from the disease are more resistant than strains from only colonized patients. See Hol, C et al. (1993) Lancet 341: 1281, Jordan, KL et al. (1990) Am. J. Med. 88 (suppl. 5A): 28S]. Serum resistance may therefore be considered to be a virulence factor of bacteria. Opsonization activity has been observed in the serum of children collected from otitis media.
Antigens targeted by these different immune responses in the human body are proteins of OMP B1, 84 kDa, whose expression is regulated by iron and recognized by the sera of patients with pneumonia. See Sethi, S. et al. (1995) Infect. Immun. 63: 1516 and UspA1 and UspA2 (Chen D. et al. (1999), Infect. Immun. 67: 1310, except for that.
Several other membrane proteins present on the surface of Moraxella catalis have been characterized by biochemical methods or when they are effectively involved in inducing protective immunity. Murphy, TF (1996) Microbiol. Rev. 60: 267]. In the mouse pneumonia model, the presence of antibodies produced against some of these proteins (UspA, CopB) makes the infection of the lungs disappear faster. Another polypeptide (OMP CD) is well conserved among Moraxella catalis strains and exhibits homology with the porins of Pseudomonas aeruginosa, which have been shown to be effective against these bacteria in animal models.
The frequency of Moraxella catalis infections has increased significantly over the past decades. This is due to the emergence of multiple antibiotic resistant strains and an increase in populations with weak immune systems. It is no longer difficult to isolate Moraxella catarrhalis strains that are resistant to some or all of the standard antibiotics. This phenomenon raises the medical need and demand for new antibiotics, vaccines, drug screening methods, and diagnostic tests for the organism.
Summary of the Invention
The present invention relates to BASB027, in particular BASB027 polypeptides and BASB027 polynucleotides, recombinants thereof and methods of producing them. In another aspect, the present invention relates to methods of using the polypeptides and polynucleotides, among others, including the prevention and treatment of diseases caused by microorganisms. In a further aspect, the invention relates to diagnostic assays for detecting diseases associated with infections by microorganisms and conditions associated with such infections, such as assays for detecting the expression or activity of BASB027 polynucleotides or polypeptides.
Various changes and modifications within the scope and spirit of the invention will be readily apparent to those skilled in the art from the detailed description set forth below and elsewhere herein.
The present invention relates to polynucleotides (hereinafter referred to as "BASB027 polynucleotide (s)", polypeptides encoded by them (hereinafter referred to as "BASB027" or "BASB027 polypeptide (s)"), recombinants thereof and the production thereof It is about a method. In another aspect, the invention relates to methods of using these polypeptides and polynucleotides, including vaccines against bacterial infections. In a further aspect, the present invention relates to diagnostic assays for detecting infection of certain pathogens.
The present invention relates to BASB027 polypeptides and polynucleotides described in detail below. In particular, the present invention relates to polypeptides and polynucleotides of BASB027 from Moraxella catalis, which are related by sequence homology to Neisseria meningitidis OMP85 outer membrane proteins. The invention relates in particular to BASB027 having nucleotide and amino acid sequences set forth in SEQ ID NO: 1 or 3 and SEQ ID NO: 2 or 4, respectively. It will be appreciated that the sequences listed below in the sequence listing represented by "DNA" represent one embodiment of the present invention, as those skilled in the art will find such sequences generally useful for polynucleotides comprising ribopolynucleotides. Because you will recognize that you can.
Polypeptide
In one aspect of the invention, the invention is a polypeptide of Moraxella catalis, herein referred to as "BASB027" and "BASB027 polypeptide," as well as biological, diagnostic, prophylactic, clinical or therapeutically useful variants thereof. And it provides a composition comprising the same.
The invention also relates to (a) at least 85% identity, preferably at least 90% identity, more preferably at least 95% identity, most preferably at least 97-99% identity with the sequence of SEQ ID NO: 2 or 4 or Isolated polypeptide comprising an amino acid sequence exhibiting correct identity; (b) at least 85% identity, preferably at least 90% identity, more preferably at least 95% identity, even more preferably with SEQ ID NO: 1 or 3 over the entire length of SEQ ID NO: 1 or 3, respectively A polypeptide encoded by an isolated polynucleotide comprising a polynucleotide sequence exhibiting 97-99% or correct identity; Or (c) at least 85% identity, preferably at least 90% identity, more preferably at least 95% identity, even more preferably at least 97-99% identity or correct with the amino acid sequence of SEQ ID NO: 2 or 4 A polypeptide encoded by an isolated polynucleotide is provided that includes a polynucleotide sequence that encodes a polypeptide that exhibits identity.
The BASB027 polypeptide provided in SEQ ID NO: 2 or 4 is BASB027 polypeptide from Moraxella catarrhalis strain MC2931 (ATCC43617).
The present invention also provides immunogenic fragments of the BASB027 polypeptide that are contiguous portions of the BASB027 polypeptide having the same or substantially identical immunogenic activity as the polypeptide comprising the amino acid sequence of SEQ ID NO: 2 or 4; That is, the fragment (if bound to the carrier, if necessary) can induce an immune response that recognizes the BASB027 polypeptide. Such immunogenic fragments can include, for example, a BASB027 polypeptide lacking an N-terminal leader sequence, and / or a transmembrane domain, and / or a C-terminal anchor domain. In a preferred aspect, the immunogenic fragment of BASB027 according to the invention is at least 85% identity, preferably at least 90% identity, more preferably at least 90% identity with the sequence of SEQ ID NO: 2 or 4 over the entire length of SEQ ID NO: 2 Comprises substantially all of the extracellular domain of a polypeptide that exhibits at least 95% identity, most preferably 97-99% identity.
A fragment is a polypeptide having an amino acid sequence which is wholly identical to some but not all of any amino acid sequence of any polypeptide of the invention. According to the BASB027 polypeptide, fragments may be "free-standing" or included in larger polypeptides in which they form part or region, most preferably in a single contiguous region in a single larger polypeptide. May be included.
Preferred fragments include a series of contiguous sequences comprising, for example, truncation polypeptides having an amino acid sequence of SEQ ID NO: 2 or 4, or a portion thereof, such as amino- and / or carboxyl-terminal amino acid sequences. It includes residues. Also preferred is a degraded form of the polypeptide of the invention produced by or in a host cell. Alpha helix and alpha helix forming region, beta sheet and beta sheet forming region, trun and turn forming region, coil and coil forming region, hydrophilic region, hydrophobic region, alpha amphipathic region, beta amphipathic region, flexible region Also preferred are fragments characterized by structural or functional appendages, such as fragments comprising surface forming regions, substrate binding regions, and high antigenicity index regions.
Another preferred fragment is an isolated polypeptide comprising an amino acid sequence having at least 15, 20, 30, 40, 50 or 100 contiguous amino acids from the amino acid sequence of SEQ ID NO: 2 or 4, or SEQ ID NO: 2 or An isolated polypeptide comprising an amino acid sequence having at least 15, 20, 30, 40, 50 or 100 consecutive amino acids truncated or deleted from the amino acid sequence of 4.
Fragments of polypeptides of the invention can be used to generate corresponding full length polypeptides by peptide synthesis: therefore, these fragments can be used as intermediates to produce full length polypeptides of the invention.
Particular preference is given to variants in which several, five to ten, one to five, one to three, one to two or one amino acid is substituted, deleted or added in any combination.
Polypeptides or immunogenic fragments of the invention may be in the form of "mature" proteins or part of larger proteins such as precursors or fusion proteins. It may often be advantageous to include additional amino acid sequences containing secretory or leader sequences, pro-sequences, sequences conducive to purification such as multiple histidine residues, or additional sequences for stability during recombinant production. It is also desirable to add exogenous polypeptides or lipid tails or polynucleotide sequences to increase the immunogenic potency of the final molecule.
In one preferred aspect, the invention is a genetically engineered construct comprising various portions of the constant regions of the heavy or light chains of the polypeptides of the invention, or fragments thereof, and immunoglobulins of various subclasses (IgG, IgM, IgA, IgE) It relates to a soluble fusion protein. Preferred as immunoglobulins are the constant portions of the heavy chain of human IgG, in particular IgG1, in which fusion is performed in the hinge region. In certain embodiments, the Fc moiety can be isolated simply by incorporating a degradation sequence that can be degraded by blood coagulation factor Xa.
The present invention also relates to methods of making these fusion proteins by genetic engineering and their use for drug screening, diagnosis and treatment. A further aspect of the invention also relates to polynucleotides encoding such fusion proteins. Examples of fusion protein techniques can be found in International Patent Applications WO94 / 29458 and WO94 / 22914.
Such proteins may be chemically conjugated or expressed as recombinant fusion proteins such that increased levels can be produced in the expression system as compared to unfused proteins. The fusion partner may assist in providing a T helper epitope (immunological fusion partner), preferably a T helper epitope recognized by humans, or in expressing a protein (expression enhancer) in higher yield than a native recombinant protein. . Preferably the fusion partner may be both an immunological fusion partner and an expression enhancing partner.
Fusion partners include protein D from Haemophilus influenza and non-structural proteins from influenza virus, NS1 (hemagglutinin). Another fusion partner is a protein known as LytA. Preferably the C terminal portion of the molecule is used. Lyta is an N-acetyl-L-alanine amidase, i.e., amidase LytA (encoded by the LytA gene (see Gene, 43 (1986) page 265-272)) (specific in peptidoglycan frameworks). It is derived from Streptococcus pneumoniae, which synthesizes autolysin) that specifically degrades binding. The C-terminal domain of LytA protein causes affinity for some choline analogs such as choline or DEAE. This property has been used to develop E. coli C-LytA expression plasmids useful for the expression of fusion proteins. Methods for purifying hybrid proteins containing C-LytA fragments at the amino terminus are described in Biotechnology; 10, (1992) pages 795-798. It is possible to use the repeating portion of the LytA molecule found at residues 178, eg, the C terminus, starting at residues 188-305.
The invention also encompasses variants of the polypeptides described above, ie, polypeptides that differ from the reference sequence by conservative amino acid sequence substitutions, wherein the residues are substituted with another residue having similar properties. Typical such substitutions are between Ala, Val, Leu and Ile; Between Ser and Thr; Between acidic residues Asp and Glu; Between Asn and Gln; Between basic residues Lys and Arg; Or between the aromatic residues Phe and Tyr.
Polypeptides of the invention can be prepared by any suitable method. Such polypeptides include isolated natural polypeptides, recombinantly produced polypeptides, synthetically produced polypeptides, or polypeptides produced by a combination of these methods. Methods of making such polypeptides are known in the art.
Most preferably, the polypeptides of the invention are derived from Moraxella catalys, but can be obtained from other organisms of the same taxonomic genus. Polypeptides of the invention can be obtained, for example, from organisms of the same taxonomic family or tree.
Polynucleotide
It is an object of the present invention to provide a polynucleotide encoding a BASB027 polypeptide, in particular a polynucleotide encoding a polypeptide designated herein as BASB027.
In a particularly preferred embodiment of the invention, the polynucleotide comprises a region encoding a BASB027 polypeptide comprising the sequence set forth in SEQ ID NO: 1 or 3 or a variant thereof comprising a full length gene.
The BASB027 polynucleotide provided in SEQ ID NOs: 1 or 3 is BASB027 polynucleotide from Moraxella catalis strain Mc2931 (ATCC43617).
In another aspect, the present invention provides, for example, BASB027 polypeptides and polynucleotides, including, for example, unprocessed RNA, ribozyme RNA, mRNA, cDNA, genomic DNA, B-DNA and Z-DNA, in particular Moraxella catalys BASB027. Isolated nucleic acid molecules are provided that encode and / or express polypeptides and polynucleotides. Additional embodiments of the present invention include biologically, diagnostically, prophylactically, clinically or therapeutically useful polynucleotides and polypeptides, and variants thereof, and compositions comprising the same.
Another aspect of the invention provides an isolated polynucleotide comprising one or more full length genes, and polynucleotides closely related thereto, encoding the BASB027 polypeptide having an estimated amino acid sequence of SEQ ID NO: 2 or 4 It is about a variant.
In another particularly preferred embodiment of the invention, the invention provides a BASB027 polypeptide from Moraxella catalyssen comprising or consisting of the amino acid sequence of SEQ ID NO: 2, 4, 6 or 8, or a variant thereof.
Using the information provided herein, such as the polynucleotide sequence set forth in SEQ ID NO: 1 or 3, the polynucleotides of the present invention encoding the BASB027 polypeptide can be prepared by standard cloning and screening methods, such as Moraxella catalis caterin as starting material ( Catlin) cells can be used to clone and sequence chromosomal DNA fragments from bacteria, followed by methods of obtaining full length clones. For example, to obtain a polynucleotide sequence of the present invention, such as the polynucleotide sequence given in SEQ ID NO: 1 or 3, typically, the chromosomal DNA of Moraxella catarrhalis catrin in E. coli or some other suitable host A library of clones is probed with radiolabeled oligonucleotides, preferably 17-mer or more oligonucleotides derived from partial sequences. Then, clones with the same DNA as the probe's DNA can be distinguished by using stringent hybridization conditions. By sequencing each clone identified by hybridization with a sequencing primer designed from the original polypeptide or polynucleotide sequence, it may be possible to extend the polynucleotide sequence in both directions to determine the full length gene sequence. have. Preferably, such sequencing is performed, for example, using modified double stranded DNA prepared from plasmid clones. Suitable techniques are described in Maniatis, T., Fritsch, E.F. and Sambrook et al., MOLECULAR CLONING, A LABORATORY MANUAL, 2nd Ed.,; Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York (1989) (see, in particular, Screening By Hybridization 1.90 and Sequencing Denatured Double-Stranded DNA Templates 13.70). In addition, direct genomic DNA sequencing can be performed to obtain full length gene sequences. To illustrate the invention, each polynucleotide described in SEQ ID NOs: 1 or 3 was found in a DNA library derived from Moraxella catarrhalis.
In addition, each DNA sequence set forth in SEQ ID NO: 1 or 3 is equivalent to the amino acid residue set forth in SEQ ID NO: 2 or 4 having an estimated molecular weight that can be calculated by using amino acid residue molecular weights known to those skilled in the art. It contains an open reading frame that encodes a protein of approximately number.
The polynucleotide of SEQ ID NO: 1 between the start codon at nucleotide number 1 and the stop codon starting at nucleotide number 2440 of SEQ ID NO: 1 encodes the polypeptide of SEQ ID NO: 2.
The polynucleotide of SEQ ID NO: 3 between the start codon at nucleotide number 1 and the stop codon starting at nucleotide number 2440 of SEQ ID NO: 3 encodes the polypeptide of SEQ ID NO: 4.
In another aspect, the invention provides (a) at least 85% identity, preferably at least 90% identity, more preferably 95 over SEQ ID NO: 1 or 3, respectively, over the entire length of SEQ ID NO: 1 or 3, respectively. Polynucleotide sequences exhibiting at least% identity, even more preferably 97-99% or exact identity; Or (b) at least 85% identity, preferably at least 90% identity, more preferably at least 95% identity, with the amino acid sequence of SEQ ID NO: 2 or 4 over the entire length of SEQ ID NO: 2 or 4, respectively, Even more preferably, an isolated polynucleotide is provided that comprises or consists of a polynucleotide sequence encoding a polypeptide that exhibits 97 to 99% identity or 100% exact identity.
Polynucleotides encoding polypeptides of the invention, including homologues and analogs from species other than Moraxella catalis, may be prepared using a suitable library of stringent hybridization conditions (eg, in the range of 45-65 ° C. and 0.1-1%). Screened with a labeled or detectable probe consisting of or comprising a sequence of SEQ ID NO: 1 or 3, or a fragment thereof; Can be obtained by a method comprising isolating a full length gene and / or genomic clone containing the polynucleotide sequence.
The present invention provides polynucleotide sequences identical to the coding sequence (open reading frame) of SEQ ID NO: 1 or 3 over the entire length. The present invention also provides the coding sequence itself for the mature polypeptide or fragment thereof, as well as another coding sequence, such as a leader or secretory sequence, pre- or pro- or prepro ( prepro) -coding sequence for a mature polypeptide or fragment thereof that is linked framed with the sequence encoding the protein sequence. Polynucleotides of the invention may also, for example, include, but are not limited to, one or more non-coding 5 'and 3' sequences, such as transcribed but not translated sequences, termination signals (eg, rho-dependent and rho- One or more non-coding sequences, including, but not limited to, independent termination signals, ribosomal binding sites, Kozak sequences, mRNA stabilizing sequences, introns, and polyadenylation signals. Polynucleotide sequences may also include additional coding sequences that encode additional amino acids. For example, marker sequences that facilitate purification of the fused polypeptide can be encoded. In certain embodiments of the invention, the marker sequence is provided in a pQE vector (Qiagen, Inc.) and described in Gentz et al., Proc. Natl. Acad. Sci., USA 86: 821-824 (1989)] or a hexa-histidine peptide, or HA peptide tag (Wild et al., Cell 37: 767 (1984)), both of which are It may be useful for purifying fused polypeptide sequences. Polypeptides of the invention also include, but are not limited to, polynucleotides comprising structural genes and their naturally bound sequences that regulate gene expression.
The nucleotide sequence encoding the BASB027 polypeptide of SEQ ID NO: 2 or 4 may be identical to the polypeptide encoding sequence contained in nucleotides 1 to 2439 of SEQ ID NO: 1 or 3, respectively. Such a sequence may also be a sequence that also encodes a polypeptide of SEQ ID NO: 2 or 4 as a result of overlapping (decrementing) the genetic code.
The term "polynucleotide encoding a polypeptide" as used herein includes a sequence encoding a polypeptide of the invention, in particular a bacterial polypeptide, more particularly a polypeptide of Moraxella catalys BASB027 having the amino acid sequence set forth in SEQ ID NO: 2 or 4 Polynucleotides. The term also refers to a single contiguous region or discontinuous region (eg, inserted phage, ie, inserted insertion sequence) that encodes a polypeptide, along with additional regions that may contain coding and / or non-coding sequences. , Polynucleotides interrupted by inserted vector sequences, inserted transposon sequences, or by RNA editing or genomic DNA reconstruction.
The invention also relates to variants of the polynucleotides described herein that encode variants of the polypeptide having the putative amino acid sequence of SEQ ID NO: 2 or 4. Fragments of the polynucleotides of the present invention can be used, for example, to synthesize full length polynucleotides of the present invention.
Another particularly preferred embodiment is that several, several, five to ten, one to five, one to three, two, one or zero amino acid residues are substituted in any combination, Polynucleotide encoding the BASB027 variant having the amino acid sequence of the BASB027 polypeptide of SEQ ID NO: 2 or 4 that is changed, deleted, and / or added. Of these, silent substitutions, additions and deletions which do not alter the properties and activity of the BASB027 polypeptide are particularly preferred.
Another preferred embodiment of the invention is a polynucleotide encoding at least 85% over its entire length with a polynucleotide encoding a BASB027 polypeptide having the amino acid sequence set forth in SEQ ID NO: 2 or 4, and a polynucleotide complementary to such polynucleotide . Also most preferred are polynucleotides comprising a region at least 90% identical in length to the polynucleotide encoding the BASB027 polypeptide and polynucleotides complementary thereto. In this regard, particular preference is given to polynucleotides that are at least 95% identical over the full length to the polynucleotides encoding the BASB027 polypeptide. Also very preferred are polynucleotides that are at least 97% identical among polynucleotides having at least 95% identity, particularly preferred are polynucleotides that are at least 99% identical with those that are at least 98% identical, and that polynucleotides having at least 99% identical Even more preferred.
Preferred embodiments are polynucleotides encoding polypeptides which possess a biological function or activity substantially the same as a mature polypeptide encoded by the DNA of SEQ ID NO: 1 or 3.
According to certain preferred embodiments of the invention, the invention provides polynucleotides which hybridize to BASB027 polynucleotide sequences, such as the polynucleotides of SEQ ID NO: 1 or 3, especially under stringent conditions.
The invention also relates to polynucleotides that hybridize to the polynucleotide sequences provided herein. In this regard, the present invention relates in particular to polynucleotides which hybridize to polynucleotides described herein under stringent conditions. As used herein, the terms “stringent condition” and “stringent hybridization condition” refer to hybridizations that occur only when there is at least 95%, preferably at least 97% identity between the sequences. Specific examples of stringent hybridization conditions include 50% formamide, 5x SSC (150 mM NaCl, 15 mM trisodium citrate), 50 mM sodium phosphate (pH7.6), 5x Denhardt's solution, 10% dextran Incubate overnight at 45 ° C. in a solution containing sulfate and 20 micrograms / ml of denatured and sheared salmon sperm DNA, followed by washing the hybridization support in 0.1 × SSC at about 65 ° C. Hybridization and washing conditions are known and are exemplified in Sambrook, et al., Molecular Cloning: A Laboratory Manual, Second Edition, Cold Spring Harbor, NY, (1989) (see, in particular, Chapter 11, supra). It is. Solution hybridization can also be used with the polynucleotide sequences provided by the present invention.
The present invention also provides a suitable library containing the complete gene for the polynucleotide sequence set forth in SEQ ID NO: 1 or 3 with the sequence of the polynucleotide sequence set forth in SEQ ID NO: 1 or 3 or a fragment thereof under stringent conditions. Screened with probe; Provided is a polynucleotide consisting of or comprising a polynucleotide sequence obtained by isolating the polynucleotide sequence. Fragments useful for obtaining such polynucleotides include, for example, probes and primers that are described in detail in any part of this application.
As described elsewhere herein in connection with the polynucleotide assay of the invention, for example, the polynucleotide of the invention may be used as a hybridization probe for RNA, cDNA and genomic DNA and to encode BASB027; Full length cDNAs can be isolated and cDNA and genomic clones of other genes with high homology, particularly high sequence identity, with the BASB027 gene. Such probes will generally comprise at least 15 nucleotide residues or base pairs. Preferably, such probes will have at least 30 nucleotide residues or base pairs and may have at least 50 nucleotide residues or base pairs. Particularly preferred probes will have at least 20 nucleotide residues or base pairs and will have fewer than 30 nucleotide residues or base pairs.
The coding region of the BASB027 gene can be isolated by screening using the DNA sequence provided in SEQ ID NO: 1 or 3 to synthesize oligonucleotide probes. Then, labeled oligonucleotides having sequences complementary to those of the genes of the present invention are used to screen libraries of cDNA, genomic DNA or mRNA to determine the members of the library in which the probe hybridizes.
Several methods are known and available to those skilled in the art for obtaining full length DNA or stretching short DNA, such as Rapid Amplication of cDNA ends (RACE) [ See Frohman, et al., PNAS USA 85: 8998-9002, 1988. Marathon ^TM (Marathon ^TM) technology latest modification of this technique, exemplified by the technology (Clontech Laboratories Inc.), for example, and significantly simplify the investigation of the longer cDNA. In Marathon ^™ technology, cDNAs are prepared from mRNA extracted from “adaptor” sequences linked to selected tissues and their respective ends. Nucleic acid amplification (PCR) is then performed to amplify the "missing" 5 'end of the DNA by using a combination of gene specific and adapter specific oligonucleotide primers. The PCR reaction is then performed on a "nested" primer, ie, a primer designed to anneal in the amplified product (adapter specific primers that typically anneal 3 'in another adapter sequence and selected gene sequences). By using a gene specific primer to anneal another 5 'in the back. The product of this reaction is then constructed either by binding the product directly to existing DNA to obtain the complete sequence or by performing a separate full length PCR using new sequence information for the design of the 5 'primer. DNA and DNA sequencing can be analyzed.
The polynucleotides and polypeptides of the invention can be used as substances and investigational reagents for the discovery of therapeutic and diagnostic agents for diseases, particularly human diseases, as described further herein, for example, in connection with polynucleotide assays. have.
Polynucleotides of the invention, which are oligonucleotides derived from the sequences of SEQ ID NO: 1 or 3, may be used in the methods described herein, but preferably are transcribed in bacteria in tissues infected with polynucleotides identified herein, in whole or in part, herein. It can be used in a method for PCR to determine whether or not. It is recognized that such sequences will be useful for diagnosing the stage of infection and form of infection that a pathogen acquires.
The present invention also provides polynucleotides encoding a mature protein and a further amino terminal or carboxy terminal amino acid, or a polypeptide that is an amino acid in the mature polypeptide (when the mature form has, for example, one or more polypeptide chains). Such sequences may play a role in the processing of proteins from precursors to mature forms, facilitate protein transport, lengthen or shorten protein half-lives, or facilitate manipulation of proteins for assay or production, among others. Can be. As is generally the case in vivo, additional amino acids can be processed from mature proteins by cellular enzymes.
For each polynucleotide and all polynucleotides of the invention, the invention provides polynucleotides complementary thereto. It is preferred that these complementary polynucleotides are completely complementary to each polynucleotide to which they are complementary.
The precursor protein with the mature form of the polypeptide fused to one or more prosequences may be an inactive form of the polypeptide. Each inert precursor is generally activated when the prosequence is removed. Some or all prosequences may be removed before activation. In general, such precursors are called proproteins.
In addition to the standard A, G, C, T / U notation for nucleotides, the term "N" may also be used to describe certain polynucleotides of the invention. "N" means that any one of the four DNA or RNA nucleotides may appear at a designated position in the DNA or RNA sequence, but N is taken with adjacent nucleotide positions, so that when read in the correct reading frame, It is preferred that it is not a nucleic acid having the effect of producing an early termination codon of.
In summary, the polynucleotides of the present invention may be directed to a mature protein, a mature protein and a leader sequence (also referred to as a preprotein), a precursor of a mature protein having one or more prosequences other than the leader sequence of the preprotein, or a proprotein. Preproproteins may be encoded that are precursors and have one or more prosequences and leader sequences that are generally removed during processing to produce active and mature forms of the polypeptide.
According to one aspect of the invention, the invention provides the use of the polynucleotide of the invention for treatment or prophylaxis, in particular for genetic immunization.
The use of the polynucleotides of the present invention in gene immunization is preferably in a suitable delivery method such as direct delivery of plasmid DNA into muscles. Wolff et al., Hum Mol Genet (1992) 1: 363, Manthorpe et al. , Hum. Gene Ther. (1983) 4: 419], delivery of DNA complexed with specific protein carriers (Wu et al., J Biol Chem. (1989) 264: 16985], co-precipitating DNA with calcium phosphate [Benvenisty & Feshef. PNAS USA, (1986) 83: 9551], encapsulation of DNA in the form of various liposomes (Kaneda et al., Science (1989) 243: 375), particle bombardment [Tang et al., Narure (1992) 356: 152, Eisenbraun et al., DNA Cell Biol (1993) 12: 791 and in vivo infection with cloned retroviral vectors (see Seeger et al., PNAS USA (1984) 81: 5849). will be.
Vector, host cell, expression system
The invention also relates to a vector comprising a polynucleotide or polynucleotides of the invention, a host cell genetically engineered with the vector of the invention and a method of producing a polypeptide of the invention by recombinant technology. Cell-free translation systems can also be used to generate proteins by using RNA derived from the DNA constructs of the invention.
Recombinant polypeptides of the invention can be prepared from genetically engineered host cells comprising expression systems by methods known to those skilled in the art. Thus, in another aspect, the present invention relates to an expression system comprising the polynucleotide or polynucleotides of the present invention, a host cell genetically engineered with the expression system, and a method of producing a polypeptide of the present invention by recombinant technology. .
In the recombinant production method of the polypeptide of the present invention, the host cell may be genetically engineered to incorporate the expression system or a part thereof or the polynucleotide of the present invention. Introduction of polynucleotides into host cells is described by Davids, et al., BASIC METHODS IN MOLECULAR BIOLOGY, (1986) AND Sambrook, et al., MOLECULAR CLONING: A LABORATORY MANUAL, 2nd Ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY (1989)], methods described in many standard experimental manuals, such as calcium phosphate transfection, DEAE-dextran mediated transfection, transfection, microinjection, cationic lipid-mediated transfection, It is performed by electroporation, transduction, scrape loading, ballistic introduction and infection.
Representative examples of suitable hosts include bacterial host cells such as streptococci, staphylococci, enterococci, Escherichia coli, Streptomyces, cyanobacteria, Bacillus Cells of Bacillus subtilis, Neisseria menididis and Moraxella cathalis; Fungal cells such as yeast, Kluveromyces, Saccharomyces, basidiomycetes, Candida albicans and Aspergillus; Animal cells such as CHO, COS, HeLa, C127, 3T3, BHK, 293, CV-1 and Bowes melanoma cells; Plant cells, such as cells of a herbaceous or pizza plant.
A wide variety of expression systems can be used to produce the polypeptides of the invention. Such vectors include, among other vectors, chromosomal-, episomal-, and virus-derived vectors, such as vectors derived from bacterial plasmids, vectors derived from bacteriophage, vectors derived from transposons, yeast episomes, among others. Derived vectors, vectors derived from insertion elements, vectors derived from yeast chromosomal elements, baculoviruses, papova viruses such as SV40, vaccinia virus, adenovirus, fowl pox viruses, Pseudorabies Vectors derived from pseudorabies viruses, picomaviruses, retroviruses, and alphaviruses, and combinations thereof, such as plasmids and bacteriophage gene elements such as cosmids and phagemids Included. Expression system constructs may contain regulatory regions that not only cause expression to occur, but also regulate. In general, any system or vector suitable for maintaining, propagating, expressing a polynucleotide and / or for expressing a polypeptide in a host can be used for expression in this regard. Appropriate DNA sequences can be inserted into expression systems by any of a variety of known and conventional techniques, such as those described in Sambrook et al., MOLECULAR CLONING, A LABORATORY MANUAL, above.
In recombinant expression systems in eukaryotic cells, when secreted translated proteins into the lumen of the endoplasmic reticulum, into the extravaginal space or into the extracellular environment, appropriate secretion signals may be incorporated into the expressed polypeptide. These signals may be exogenous or heterologous to the polypeptide.
Polypeptides of the invention are known methods including ammonium sulfate or ethanol precipitation, acid extraction, anion or cation exchange chromatography, phosphocellulose chromatography, hydrophobic interaction chromatography, hydrophilic chromatography, hydroxyapatite chromatography and lecithin chromatography Can be harvested and purified from recombinant cell culture. Most preferably, iron metal affinity chromatography (IMAC) is used for purification. Known techniques for refolding proteins can be used to regenerate the active form when the polypeptide is denatured during intracellular synthesis, isolation and / or purification.
The expression system can be a recombinant living microorganism such as a virus or a bacterium. The gene of interest may be inserted into the genome of a live recombinant virus or bacterium. Inoculation and in vivo infection with such live vectors will lead to in vivo expression of the antigen and induction of an immune response. Viruses and bacteria used for this purpose include, for example, poxviruses (e.g. vaccinina, foulpox, canarypox), alphaviruses (Sindbis virus Semliki Forest Virus). ), Venezuelan Equine Encephalitis Virus, Adenovirus, Adeno-associated virus, Piconavirus (Poliovirus, Renovirus), Herpes virus (Varicella zoster virus, etc.), Listeria , Salmonella, Shigella, BCG These viruses and bacteria are toxic or can be attenuated in various ways to obtain a live vaccine, which live vaccines also form part of the invention.
Diagnostic, prognosis, serotyping and mutation assays
The invention also relates to the use of the BASB027 polynucleotides and polypeptides of the invention for use as diagnostic reagents. Detection of BASB027 polynucleotides and / or polypeptides in eukaryotic cells, in particular mammals, in particular in humans, provides diagnostic methods for diagnosing a disease, staging of a disease, or in response to an infecting organism to a drug. Those subjects susceptible to infection or susceptible to eukaryotic cells, preferably mammals and in particular humans, in particular organisms comprising the BASB027 gene or protein, are not only described by the methods described herein, but also by various known techniques. Or at the amino acid level.
Polypeptides and polynucleotides for prognosis, diagnostic or other analysis can be obtained from substances from the body of a putatively infected and / or infected individual. Polynucleotides, especially DNA or RNA, from any of these sources can be used directly for detection or enzymatically amplified by using PCR or any amplification technique prior to analysis. RNA, in particular mRNA, cDNA and genomic DNA, can also be used in the same way. Using amplification, characterizing the species and strain of an infection or endogenous organism present in a subject can be performed by analysis of the genotype of a given polynucleotide of the organism. Deletion and insertion can be detected by changing the size of the amplified product relative to the genotype of the reference organism selected from the relevant organism, preferably from different species of the same genera or from different strains of the same species. Point mutations can be identified by hybridizing amplified DNA to labeled BASB027 polynucleotide sequences. Perfectly or significantly matched sequences can be distinguished from incompletely or more significantly mismatched duplexes by DNase or RNase digestion on DNA or RNA, or by detecting differences in melting temperature or restoration kinetics, respectively. have. Differences in polynucleotide sequences can also be detected by changes in the electrophoretic mobility of the polynucleotide fragments in the gel compared to the reference sequence. Such testing may be performed with or without a denaturing agent. Polynucleotide differences can also be detected by direct DNA or RNA sequencing (Myers et al., Science, 230: 1242 (1985)). Sequence changes at specific positions can also be revealed by nuclease protection assays such as RNase, V1 and S1 protection assays or chemical degradation methods. Cotton et al., Proc. Natl. Acad. Sci., USA, 85: 4379-4401 (1985).
In another embodiment, an array of oligonucleotide probes comprising a BASB027 nucleotide sequence or fragment thereof can be configured to perform efficient screening of, for example, gene mutations, serotypes, taxonomic classification or identification. Array methods are well known, have general applicability, and can be used to solve a variety of problems in the field of molecular genetics, including gene expression, gene binding and gene variability. Chee et al., Science, 274: 610 (1996).
Thus, in another embodiment, the present invention,
(a) a polynucleotide of the invention, preferably the nucleotide sequence of SEQ ID NO: 1 or 3 or a fragment thereof;
(b) a nucleotide sequence complementary to the sequence of (a);
(c) a polypeptide of the invention, preferably the polypeptide of SEQ ID NO: 2 or 4 or a fragment thereof; or
(d) relates to a diagnostic kit comprising an antibody against a polypeptide of the invention, preferably the polypeptide of SEQ ID NO: 2 or 4.
In any of the above kits, it will be appreciated that (a), (b), (c) or (d) may comprise substantial components. Such kits will be useful for diagnosing disease or sensitivity to disease, among other things.
The invention also relates to the use of the polynucleotides of the invention as diagnostic reagents. Detection of a mutated form of SEQ ID NO: 1 or 3 in connection with a polynucleotide, preferably a disease or pathogenicity, of the present invention can be used to diagnose a disease, prognosis of a disease process, measure a disease stage, or underexpression of a polynucleotide. It provides a diagnostic tool that can be added to measure sensitivity to diseases that induce overexpression or altered expression. Organisms with mutants of polynucleotides, preferably infectious organisms, can be detected at the polynucleotide level by various techniques, such as those described elsewhere herein.
Cells from organisms having mutations or polymorphisms (allelic variants) of polynucleotides and / or polypeptides of the invention can be detected at the polynucleotide or polypeptide level to enable sera by various techniques. For example, RT-PCR can be used to detect mutations in RNA. Particular preference is given to using RT-PCR with an automated detection system, for example GeneScan. RNA, cDNA or genomic DNA may also be used for the same purpose, ie PCR. For example, PCR primers complementary to polynucleotides encoding the BASB027 polypeptide can be used to identify and analyze mutants.
The invention also provides primers in which 1, 2, 3 or 4 nucleotides have been removed from the 5 'and / or 3' ends. These primers can be used to amplify BASB027 DNA and / or RNA isolated from a sample derived from a subject, such as a substance obtained from the body, among other things. Such primers can be used to amplify polynucleotides isolated from an infected subject, so that the polynucleotides can be given to various techniques for identifying polynucleotide sequences. In this method, mutations in the polynucleotide sequence can be detected and used to diagnose and / or prognose an infection or its steps or processes, or to serotype and / or classify an infectious agent.
The invention also includes measuring increased expression levels of polynucleotides having a sequence of SEQ ID NO: 1 or 3 from a sample derived from a subject, such as a substance obtained from the body, for example, a disease, preferably a bacterial infection More preferably, the present invention provides a method for diagnosing an infection caused by Moraxella catarrhalis. Increased or decreased expression of BASB027 polynucleotides may be any of the methods known in the art for quantification of polynucleotides, such as amplification, PCR, RT-PCR, RNase protection, Northern blotting, spectroscopy. It can be measured by using analysis and other hybridization methods.
In addition, a diagnostic assay according to the invention that detects overexpression of the BASB027 polypeptide compared to normal regulatory tissue samples can be used, for example, to detect the presence of an infection. Assay techniques that can be used to measure the levels of BASB027 polypeptide in samples derived from the host, such as materials from the body, are known to those skilled in the art. Such assay methods include radioimmunoassay, competitive binding assay, Western blot analysis, antibody sandwich assay, antibody detection and ELISA assay.
The polynucleotides of the present invention may be used as components of polynucleotide arrays, preferably high density arrays or grids. These high density arrays are particularly useful for diagnostic and prognostic purposes. For example, a set of spots comprising different genes and further comprising polynucleotides or polynucleotides of the invention may be hybridized using probes obtained or derived from, for example, a sample obtained from the body, or Probing by using nucleic acid amplification can be used to determine the presence of a specific polynucleotide sequence or related sequence in a subject. Such presence may indicate the presence of a pathogen, in particular the presence of Moraxella catalis, and may be useful for diagnosing and / or prognosticing a disease or course of a disease. Preference is given to a grid comprising many variants of the polynucleotide sequence of SEQ ID NO: 1 or 3. It is also preferred to include many variants of the polynucleotide sequence encoding the polypeptide sequence of SEQ ID NO: 2 or 4.
Antibodies
Polypeptides and polynucleotides or variants thereof of the invention, or cells expressing them, can be used as immunogens to produce antibodies specific for such polypeptides or polynucleotides, respectively.
In certain preferred embodiments of the invention, the invention provides an antibody against a BASB027 polypeptide or polynucleotide.
Antibodies generated against a polypeptide or polynucleotide of the invention may comprise an epitope-containing fragment of one or both of the polypeptides and / or polynucleotides of the invention, one or both analogs thereof, or one or two of them. Cells expressing all can be obtained by administering to an animal, preferably an animal other than a human, using a conventional protocol. In the case of producing monoclonal antibodies, techniques known in the art can be used which provide antibodies produced by continuous cell line cultures. Examples of such techniques are described in Kohler, G. and Milstein, C., Nature 256: 495-497 (1975); Kozbor et al., Immunology Today 4: 72 (1983); Cole et al., Pg. 77-96, MONOCLONAL ANTIBODIES AND CANCER THERAPY, Alan R. Liss, Inc. (1985).
Techniques for the production of single chain antibodies (US Pat. No. 4,946,778) can be adapted to generate single chain antibodies against the polypeptides or polynucleotides of the invention. In addition, transformed mice or other organisms or animals, such as other mammals, can be used to express humanized antibodies immunospecific to the polypeptides or polynucleotides of the invention.
In addition, phage display technology can be used to select antibody genes with binding activity to polypeptides of the invention from a repertoire of PCR amplified v-genes from humans screened with anti-BASB027 or from native libraries [ See, McCafferty, et al., (1990), Nature 348, 552-554; Marks et al., (1992) Biotechnology 10, 779-783. The affinity of these antibodies can be improved, for example, by chain shuffling (Clackson et al., (1991) Nature 352: 628).
The antibodies described above can be used to isolate and identify clones expressing polypeptides or polynucleotides of the invention, for example, to purify the polypeptides or polynucleotides by affinity chromatography.
Thus, among other things, antibodies against BASB027 polypeptide or BASB027 polynucleotide can be used to treat infections, in particular bacterial infections.
Polypeptide variants include antigenic, epitope or immunologically equivalent variants that form certain aspects of the invention.
Preferably, the antibody or variant thereof is changed to be less immunogenic in the subject. For example, when the subject is a human, the antibody is most preferably “humanized” and the complementarity determining region or region (s) of the hybridoma derived antibody is described, for example, in Jones et al. (1986), Nature 321, 522-525 or Tempest et al., (1991) Biotechnology 9, 266-273, to be incorporated into human monoclonal antibodies.
Antagonists and Agents-Assays and Molecules
Polypeptides and polynucleotides of the invention can also be used to assess the binding of small molecule substrates and ligands, for example, in cells, cell-free preparations, chemical libraries, and naturally occurring mixtures. These substrates and ligands can be natural ligands and ligands or structural or functional agents (Coligan et al., Current Protocols in Immunology 1 (2): Chapter 5 (1991)).
Screening methods can simply measure the binding of a test compound to a polypeptide or polynucleotide, or to a cell or membrane carrying the polypeptide or polynucleotide, or to a fusion protein of the polypeptide by means of a label directly or indirectly associated with the test compound. have. In addition, the screening method may include a competition reaction with a labeled competitor. In addition, these screening methods can be tested by using a detection system appropriate for cells comprising a polypeptide or polynucleotide whether the test compound produces a signal produced by activation or inhibition of the polypeptide or polynucleotide. Inhibitors of activation are generally assayed in the presence of known agents, and effects on activation by agents by the presence of test compounds are observed. Constitutively active polypeptides and / or constitutively expressed polypeptides and polynucleotides, if possible, in the absence of an agonist or inhibitor, test, if possible, whether the test compound inhibits the activation of the polypeptide or polynucleotide. Can be used in screening methods. In addition, the screening method simply mixes the test compound with a solution containing the polypeptide or polynucleotide of the present invention to form a mixture, to determine the BASB027 polypeptide and / or polynucleotide activity in the mixture, to determine the BASB027 polypeptide and And / or comparing the polynucleotide activity with standard activity. Fusion proteins, such as those made from the Fc moiety and the BASB027 polypeptide, as described herein, can also be used in high yield screening assays to identify antagonists of polypeptides of the invention, as well as phylogenetic and / or functionally related polypeptides. D. Bennett et al., J Mol Recognition, 8: 52-58 (1995); and K. Johanson et al., J Biol Chem, 270 (16): 9459-9471 (1995).
Polynucleotides, polypeptides, and antibodies that bind and / or interact with polypeptides of the invention can also be used to construct screening methods to detect the effect of added compounds on the production of mRNA and / or polypeptides in cells. For example, ELISA assays can be constructed to measure secreted or cell related levels of polypeptides using monoclonal and polyclonal antibodies by standard methods known in the art. Such assays can be used to find agents (also referred to as antagonists or agents, respectively) that can inhibit or enhance the production of polypeptides from suitably engineered cells or tissues.
The invention also provides a method for screening a compound to identify a compound that enhances (agents) or blocks (antagonists) the action of a BASB027 polypeptide or polynucleotide, in particular a bactericidal and / or bactericidal polypeptide or polynucleotide. Such screening methods may include high yield techniques. For example, to screen for an agonist or antagonist, a synthetic reaction mixture, cell compartment, such as a membrane, cell envelope or cell wall, or formulation thereof comprising a BASB027 polypeptide and a labeled substrate or ligand thereof, may be used as a BASB027 agonist or antagonist. Culture in the absence or presence of a test molecule which may be. The ability of candidate molecules to enhance and inhibit BASB027 polypeptides reflects reduced binding of labeled ligands or reduced production of products from such substrates. The molecules that bind free, ie without inducing the effects of the BASB027 polypeptide, appear to be mostly good antagonists. Molecules that bind well and, if desired, increase product yield from the substrate, increase signal transduction, or increase chemical channel activity are agents. If desired, production of the product from the substrate, signal transduction, or detection of the rate or level of chemical channel activity can be enhanced by using a reporter system. Reporter systems that may be useful in this regard include, but are not limited to, reporter genes that are responsive to changes in colorimetric labeled substrates, BASB027 polynucleotides, or polypeptide activity converted to products, and binding assays are known in the art. Known in
Another example of an assay for a BASB027 agent is a competitive assay that mixes BASB027 and a potential agent with a BASB027 binding molecule, a recombinant BASB027 binding molecule, a natural substrate or ligand, or a substrate or ligand agent under appropriate conditions for competition inhibition assays. have. BASB027 is labeled, for example, by radioactive or colorimetric compounds, allowing the number of BASB027 molecules bound or converted to a product to be accurately measured to assess the efficacy of a potential antagonist.
Potential antagonists include, among others, small organic molecules, peptides, polypeptides and antibodies that bind to and inhibit or extinguish the activity or expression of polynucleotides and / or polypeptides of the invention. Potential antagonists are also small organic molecules, peptides, polypeptides, such as closely related proteins or antibodies, that bind to the same site on a binding molecule without inducing BASB027 induced activity, thereby preventing the BASB027 polypeptide and / or polynucleotides from the binding. By exclusion, the action or expression of BASB027 polypeptide and / or polynucleotide can be inhibited.
Potential antagonists inhibit normal biological activity by preventing binding to cell binding molecules, including small molecules that bind to or fill the binding site of a polypeptide. Examples of small molecules include, but are not limited to, small organic molecules, peptides or peptide like molecules. Other potent antagonists include antisense molecules (Okano, J. Neurochem, 56: 560 (1991); OLIGODEOXYNUCLEOTIDES AS ANTISENSE INHIBITORS OF GENE EXPRESSION, CRC Press, Boca Raton, FL (1988), for a description of these molecules]. Preferred agonist antagonists include variants of BASB027 and compounds related thereto.
In another aspect of the invention, the invention is a genetically engineered comprising a variable portion of the constant regions of heavy or light chains of immunoglobulins of various subclasses (IgG, IgM, IgA, IgE), and polypeptides or fragments thereof of the invention It relates to a soluble fusion protein. As immunoglobulins, the constant portion of the light chain of human IgG, in particular IgG1, in which fusion is performed in the hinge region is preferred. In certain embodiments, the Fc moiety can be simply removed by incorporation of a degradation sequence that can be degraded by blood coagulation factor Xa. The invention also relates to methods of making these fusion proteins by genetic engineering and their use for drug screening, diagnosis and treatment. Another aspect of the invention also relates to polynucleotides encoding such fusion proteins. Examples of fusion protein techniques can be found in International Patent Applications WO94 / 29458 and WO94 / 22914.
Each of the polynucleotide sequences provided herein can be used for the discovery and development of antimicrobial compounds. Proteins encoded upon expression can be used as targets for the screening of antimicrobial drugs. In addition, a polynucleotide sequence encoding the amino terminal region of the encoded protein or Shine-Delgarno or a translation facilitating sequence of each mRNA can be used to construct the antisense to regulate the expression of the desired coding sequence. .
The invention also provides the use of a polypeptide, polynucleotide, agonist or antagonist of the invention which inhibits the initial physical interaction between a pathogen or pathogen (s) and a mammalian host involved in the eukaryotic, preferably aftermath of infection. do. In particular, the molecules of the present invention are used to inhibit adhesion of bacteria, in particular Gram negative positive and / or Gram negative bacteria, to eukaryotic, preferably mammalian, extracellular matrix proteins on endogenous devices; Inhibit bacterial adhesion between eukaryotic, preferably mammalian, extracellular matrix proteins and bacterial BASB027 proteins that mediate tissue damage; In addition to the insertion of an embedded device or other surgical technique, it can be used to block the progression of normal onset in the disclosed infection.
According to another aspect of the invention, the present invention provides BASB027 agonists and antagonists, preferably bacteriostatic or bactericidal agents and antagonists.
Antagonists and agents of the invention can be used, for example, to prevent, inhibit and / or treat diseases.
In another aspect, the present invention relates to mimotope of the polypeptide of the present invention. Mimotops are sufficiently similar (sequentially or structurally) to the original peptide, which can be recognized by an antibody that recognizes the original peptide, or can produce an antibody that recognizes the original peptide when combined with a suitable carrier. Peptide sequence.
Peptide mimotopes can be designed for specific purposes by addition, deletion or substitution of the resulting amino acids. Thus, peptides can be modified to facilitate conjugation to protein carriers. For example, it may be desirable for certain chemical conjugation methods to include terminal cysteines. In addition, the peptide conjugated to the protein carrier includes a hydrophobic end behind the conjugated end of the peptide such that the free unconjugated end of the peptide is maintained in association with the surface of the carrier protein, thereby allowing the entire original molecule The peptide is present in a form that closely resembles the form of the peptide as found in the composition of. For example, the peptide can be modified to have a tail with N-terminal cysteine and C-terminal hydrophobic amide. In addition, addition or substitution of the D-stereoisomers of one or more amino acids can be performed to produce derivatives that are beneficial, for example, to enhance the stability of the peptide.
Peptide mimotops can also be identified by using antibodies that can bind to polypeptides of the invention by using techniques such as phage display technology (EP 0 552 267 B1). This technique mimics the structure of the original peptide and, thus, can bind to anti-native peptide antibodies, but they themselves produce large amounts of peptide sequences that do not necessarily share significant sequence homology to the original polypeptide. Create
vaccine
Another aspect of the invention provides a BASB027 polynucleotide and / or polypeptide, or sufficient to generate an antibody and / or T cell immune response that protects a subject from infection, particularly bacterial infections and most particularly Moraxella catarrhal infections, or A method for inducing an immunological response in a subject, particularly a mammal, preferably a human body, including inoculating the subject with a fragment or variant thereof. Also provided are methods by which immunological responses mitigate bacterial replication. Another aspect of the invention is a method of inducing an immunological response in a subject, wherein the nucleic acid vector, sequence or ribosome is delivered to the subject to thereby determine whether the disease is present in the subject, BASB027 polynucleotides, preferably to induce an immunological response, such as to generate an antibody and / or T cell immune response comprising, for example, cytokine-producing T cells or cytotoxic T cells, to protect a human And / or inducing a BASB027 polynucleotide and / or polypeptide, or fragment or variant thereof, for in vivo production of the polypeptide, or fragment or variant thereof. One example of administering a gene is to promote the gene into cells that are required for coating on particles and the like. Such nucleic acid vectors may include DNA, RNA, ribozymes, altered nucleic acids, DNA / RNA hybrids, DNA-protein complexes or RNA-protein complexes.
Another aspect of the invention is an BASB027 polynucleotide and / or as an immunological composition, when introduced into a subject, preferably a subject capable of exhibiting an immunological response induced in a human, preferably a human. Thereby inducing an immunological response to the encoded polypeptide, comprising recombinant BASB027 polynucleotide and / or polypeptide encoded therefrom, and / or said BASB027 polynucleotide of the invention, polypeptide encoded therefrom, or An immunological composition comprising DNA and / or RNA encoding and expressing an antigen of an external polypeptide. The immunological response can be used for therapeutic or prophylactic purposes and can take the form of cellular immunity and / or antibody immunity such as cellular immunity generated from CTL or CD4 + T cells.
The BASB027 polypeptide or fragment thereof cannot produce or produce antibodies on its own, but will produce a fused or altered protein that may stabilize the first protein and have antigenic and / or immunogenicity, and preferably protective properties. Can be fused with a co-protein or chemical moiety. Thus, the fused recombinant protein preferably comprises an antigenic co-protein, or protein, such as lipoprotein D from Haemophilus influenza, glutathione-S-transferase (GST) or beta-calactosidase. It further includes any other relatively large co-protein that stabilizes and facilitates its production and purification. Co-proteins can also serve as adjuvants in that they provide generalized stimulation of the immune system of the organism that contains the protein. Such co-proteins may be linked to either the amino- or carboxy-terminus of the first protein.
The present invention provides a composition, preferably a vaccine composition, comprising a polypeptide and / or polynucleotide of the invention and an immunostimulatory DNA sequence as described in Sato, Y. et al., Science 273: 352 (1996), And methods.
In addition, the present invention, together with Moraxella catarrhalis, requires polynucleotides or their specific fragments known to encode non-variable regions of bacterial cell surface proteins in polynucleotide constructs used in gene immunization experiments in animal infection models. Provides a way to use it. Such experiments will be particularly useful for identifying protein epitopes that can elicit a prophylactic or therapeutic immune response. This study is a monoclonal for specific purposes derived from essential organs in animals resistant to or free of infection, for the development of a prophylactic or therapeutic agent for bacterial infections in mammals, in particular the human body, in particular Moraxella catarrhal infections. It will enable the preparation of antibodies.
The present invention also encompasses vaccine formulations comprising the immunogenic recombinant polypeptides and / or polynucleotides of the invention together with a suitable carrier, such as a pharmaceutically acceptable carrier. Since polypeptides and polynucleotides can be destroyed in the stomach, each of these is preferably administered by parenteral administration, including, for example, subcutaneously, intramuscularly, intravenously or intradermally. Formulations suitable for parenteral administration include aqueous and non-aqueous sterile injectable solutions which may contain oxidation inhibitors, buffers, bacteriostatic compounds, and solutes that render the formulation isotonic with the body fluids of the subject, preferably blood; And aqueous and non-aqueous sterile suspensions which may include suspending agents or thickening agents. Such formulations may be present in single or multiple dose containers, such as sealed ampoules and vials, and may be stored in a lyophilized state requiring the addition of a sterile liquid carrier just prior to use.
Vaccine formulations of the present invention may also include adjuvant systems that enhance the immunogenicity of the formulations. Preferably the auxiliary system preferentially increases the response of the TH1 type.
Immune responses can be broadly classified into two extreme classes: humoral immune responses or cell mediated immune responses (typically characterized by antibody and cell effector mechanisms, respectively). This kind of response is called TH1-type response (cell-mediated response), and TH2-type immune response (humoral response).
Extreme TH1-type immune responses can be characterized by the generation of antigen specific, haplotype restricted cytotoxic T lymphocytes, and natural killer cell responses. In mice, TH1-type responses are often characterized by the production of antibodies of the IgG2a subtype, but in the human body these responses correspond to IgG1 type antibodies. TH2-type immune responses are characterized by the generation of a broad range of immunoglobulin isotypes, including IgG1, IgA, and IgM in mice.
It can be understood that the driving force behind the development of these two types of immune responses is the cytokine. High levels of TH1-type cytokines tend to favor the induction of cell-mediated immune responses against a given antibody, while high levels of TH2-type cytokines tend to favor the induction of humoral immune responses to antigens. .
The distinction between TH1 and TH2-type immune responses is not absolute. In fact, a subject can support an immune response that is described as being predominantly TH1 or predominantly TH2. However, Mosmann, T.R. and Coffman, R. L. (1989) TH1 and TH2 cells: different patterns fo lymphokine secretion lead to different functional properties, Annual review of Immunology, 7, p145-173. Often it is easy. Typically, the TH1-type response is associated with INF-γ and IL-2 cytokines by T-lymphocytes. Other cytokines often directly involved in the induction of TH1-type immune responses are not produced by T-cells such as IL-12. In contrast, TH2-type responses are associated with the secretion of IL-4, IL-5, IL-6 and IL-13.
It is known that certain vaccine adjuvants are particularly suitable for stimulation of TH1 or TH2-type cytokine responses. Typically, the best indicator of the TH1: TH2 balance of the immune response after vaccination or infection is a direct measurement of the production of TH1 or TH2 cytokines by T lymphocytes in vitro after restimulation with antigen, and / or antigen specific. Measurement of the IgG1: IgG2a ratio of the antibody response.
Thus, TH1-type adjuvants stimulate high levels of TH1-type cytokines by stimulating isolated T-cell populations when re-stimulated with in vitro antigens, and antigen-specific immunoglobulin responses and CD8 + associated with TH1-type isotypes. It is an adjuvant that promotes the development of both cytotoxic T lymphocytes.
Adjuvants capable of preferentially stimulating the TH1 cell response are described in International Patent Applications WO94 / 00153 and WO95 / 17209.
3 De-O-acylated monophosphoryl lipid A (3D-MPL) is one such adjuvant. Such adjuvants are known from GB 2220211 (Ribi). Chemically, this adjuvant is a mixture with 4, 5 or 6 acylated chains of 3 De-O-acylated monophosphoryl lipid A, and was prepared by Bibi Immunochem, Montana. Are manufactured. A preferred form of De-O-acylated monophosphoryl lipid A is disclosed in European Patent No. 0 689 454 B1 (SmithKline Beecham Biologicals SA).
Preferably, the particles of 3D-MPL are small enough to be aseptically filtered through a 0.22 micron membrane (European Patent No. 0 689 454).
3D-MPL is present in the range of 10 μg to 100 μg per dose, preferably in the range of 25 to 50 μg, wherein the antigen is typically present at 2 to 50 μg per dose.
Another preferred adjuvant includes the Hplc purified non-toxic fraction derived from the bark of QS21, Quillaja Saponaria Molina.
The production method of QS21 is disclosed in US Pat. No. 5,057,540.
Non-reaction producing co-formulations containing QS21 are described above (WO 96/33739). Such formulations comprising QS21 and cholesterol have been found to be successful TH1 stimulation aids when formulated with antigen.
Another adjuvant that is a preferential stimulator of TH1 cell response includes immunoregulatory oligonucleotides, eg, unmethylated CpG sequences as disclosed in WO 96/02555.
Combinations of different TH1 stimulatory adjuvants, such as those described above, are also believed to provide adjuvants that are preferential stimulators of TH1 cell responses. For example, QS21 can be formulated with 3D-MPL. The ratio of QS21: 3D-MPL is typically from 1:10 to 10: 1; Preferably 1: 5 to 5: 1; Most practically 1: 1. The preferred range for optimal synergy is 3D-MPL: QS21 from 2.5: 1 to 1: 1.
Preferably, the carrier is also present in the vaccine composition according to the invention. The carrier may be an oil-in-water emulsion, or an aluminum salt, such as aluminum phosphate or aluminum hydroxide.
Preferred oil-in-water emulsions include metabolizable oils such as squalene, alpha tocopherol and Tween 80. In a particularly preferred aspect the antigen in the vaccine composition according to the invention is mixed with QS21 and 3D-MPL in said emulsion. In addition, oil-in-water emulsions may contain span 85 and / or lecithin and / or tricapryline.
Typically, for human administration, QS21 and 3D-MPL will be present in the vaccine in the range of 1 μg to 200 μg, such as 10 to 100 μg, preferably 10 to 50 μg per dose. Typically, oil-in-water will comprise 2-10% squalene, 2-10% alpha tocopherol, and 0.3-3% Tween 80. Preferably the ratio of squalene: alpha tocopherol is 1 or less, which ratio provides a more stable emulsion. Span 85 may also be present at a level of 1%. In some cases, it will be advantageous for the vaccines of the present invention to further contain stabilizers.
Non-toxic oil-in-water emulsions preferably contain a non-toxic oil, such as a scalan or squalene, an emulsifier, such as Tween 80, in an aqueous carrier. The aqueous carrier can be, for example, phosphate buffered saline.
Particularly effective adjuvant formulations comprising QS21, 3D-MPL and tocopherol in oil-in-water emulsions are described in WO 95/17210.
The invention also provides a multivalent vaccine composition comprising the vaccine formulation of the invention together with other antigens, in particular antigens useful for treating cancer, autoimmune diseases and related diseases. Such multivalent vaccine compositions may comprise a TH1 induction adjuvant as described above.
Although the present invention has been described with reference to certain BASB027 polypeptides and polynucleotides, the present invention relates to natural polypeptides and polynucleotides having additions, deletions or substitutions that do not substantially affect the immunogenicity of the recombinant polypeptide or polynucleotide, and similar It will be understood that it includes fragments of polypeptides and polynucleotides.
Compositions, Kits, and Administration
In a further aspect of the invention, the invention provides a composition comprising a BASB027 polynucleotide and / or a BASB027 polypeptide for administration to a cell or multicellular organism.
The invention also relates to compositions comprising the polynucleotides and / or polypeptides or agents or antagonists thereof discussed herein. Polypeptides and polynucleotides of the present invention may be used with unsterile or sterile carriers for use with a cell, tissue or organism, such as a pharmaceutical carrier suitable for administration to a subject. Such compositions include, for example, media additives or therapeutically effective amounts of the polypeptides and / or polynucleotides of the invention and pharmaceutically acceptable carriers or excipients. Such carriers may include, but are not limited to, saline, buffered saline, dextrose, water, glycerol, ethanol and combinations thereof. The formulation should be suitable for the mode of administration. The invention also relates to diagnostic and pharmaceutical kits comprising one or more containers filled with one or more components of the compositions of the invention described above.
Polypeptides, polynucleotides and other compounds of the invention may be used alone or in combination with other compounds, such as therapeutic compounds.
Pharmaceutical compositions can be administered in an effective and convenient manner including, for example, by topical, oral, anal, vaginal, intravenous, intraperitoneal, intramuscular, subcutaneous, intranasal, or intradermal routes.
As a therapeutic or prophylactic method, the active agent can be administered to the subject as an injectable composition, such as sterile water, preferably isotonic solution.
In a further aspect, the invention provides a therapeutically effective amount of a polypeptide and / or polynucleotide (such as a soluble form of the polypeptide and / or polynucleotide of the invention), agonist or antagonist peptide, together with a pharmaceutically acceptable carrier or excipient It provides a pharmaceutical composition comprising a low molecular weight compound. Such carriers include, but are not limited to, saline, buffered saline, dextrose, water, glycerol, ethanol and mixtures thereof. The invention also relates to pharmaceutical packs and kits comprising one or more containers filled with one or more components of the aforementioned compositions of the invention.
Polypeptides, polynucleotides and other compounds of the invention may be used alone or in combination with other compounds such as therapeutic compounds.
The composition can be used to suit the route of administration, such as the systemic or oral route. Preferred forms of systemic administration are typically infusions by intravenous injection. Other infusion routes may be used, such as subcutaneously, intramuscularly, or intraperitoneally. Other methods for systemic administration include transmucosal and transdermal administration using penetrants such as bile salts, fusidic acid or other detergents. In addition, oral administration is also possible if the polypeptides or other compounds of the invention can be formulated in an enteric or encapsulated formulation. In addition, the administration of these compounds may be ointment, and may occur locally and / or locally in the form of gels, solutions, powders and the like.
For administration to mammals, especially humans, the daily dose of the active agent is expected to be 0.1 mg / kg to 10 mg / kg, typically about 1 mg / kg. In any case, the physician will determine the most practical dose that best suits the individual and depends on the age, weight and response of the particular individual. The dosage is an example of an average case. Of course, there may be examples in which a higher or lower dose range may be advantageous for some individuals, which is within the scope of the present invention.
The dosage range required depends on the choice of peptide, the route of administration, the nature of the formulation, the propensity of the subject's condition and the judgment of the attending physician. However, suitable doses range from 0.1 to 100 μg / kg of body weight (kg) of the subject.
Vaccine compositions are conveniently in injectable form. Conventional adjuvants may be used to enhance the immune response. Suitable unit doses for the vaccine are from 0.5 to 5 μg per kg of antigen, with doses preferably administered once to three times at intervals of one to three weeks. If using the specified dose range, no deleterious toxicological effects will be observed, otherwise it should not be administered to the appropriate subject.
However, various changes in the necessary dose can be expected in terms of the variety of compounds employed and the different efficacy of the various routes of administration. For example, oral administration is expected to require higher doses than administration by intravenous injection. Since this change in capacity level is well understood in the art, it can be adjusted using standard experiments for optimization.
Sequence database, sequences and algorithms in tangible media
Polynucleotide and polypeptide sequences form useful sources of information for further identifying sequences with similar homology as well as for determining their two and three dimensional structures. This method is very easy to store sequences in a computer readable medium and then search the sequence database using data stored in known macromolecular rescue programs or using well known search tools such as GCG program packages.
The present invention also provides methods for analyzing character sequences or strings, in particular gene sequences or encoded protein sequences. For example, preferred methods of sequence analysis include identity and similarity analysis, DNA, RNA and protein structure analysis, sequence assembly, branching analysis, sequence motif analysis, open reading frame determination, nucleic acid base calling, codon usage analysis, There are methods of sequence homology such as nucleic acid base trimming, and chromatogram peak analysis for sequencing.
Computer-based methods are used to perform homology checks. The method comprises providing a first polynucleotide sequence comprising the polynucleotide sequence of the present invention in a computer readable medium; And comparing the first polynucleotide sequence with at least one second polynucleotide or polypeptide sequence to confirm homology.
Also provided is a computer-based method for performing homology confirmation, the method comprising providing a first polypeptide sequence comprising the sequence of a polypeptide of the invention in a computer readable medium and using the first polypeptide sequence. Identifying homology compared to at least one second polynucleotide or polypeptide sequence.
All publications and references, including, but not limited to, the patents and patent applications cited herein, are hereby incorporated by reference in their entirety and as specifically and individually so as to be incorporated by reference as if each publication or reference were fully published. The whole is quoted by reference. In addition, all patent applications to which this application claims priority are hereby incorporated by reference in their entirety in a manner as described above for publications and references.
Justice
"Identity" is, as is known in the art, a relationship between two or more polypeptide sequences or two or more polynucleotide sequences, as the case may be determined by comparing the sequences. In the art, “identity” also means the degree of sequence relatedness between polypeptide or polynucleotide sequences, as the case may be, determined by a match between strings of sequences. “Identity” can be readily calculated by known methods, including but not limited to those described in the following references: Computational Molecular Biology, Lesk, AM, ed., Oxford University Press, New York, 1988; Biocomputing: Informatics and Genome Projects, Smith, D. W., ed., Academic Press, New York, 1993; Computer Analysis of Sequence Data, Part I, Griffin, A.M., and Griffin, H.G., eds., Humana Press. New Jersey, 1994; Sequence Analysis in Molecular Biology, von Heine, G., Academic Press. 1987; and Sequence Analysis Primer, Gribskov, M. and Devereux, J., eds., M Stockton Press, New York; 1991; and Carillo, H., and Lipman, D., SIAM J. Applied Math., 48: 1073 (1988). The method of determining identity is designed to provide the maximum match between the sequences tested. Moreover, the method of determining identity is codified in publicly available computer programs. Computer programs that determine the identity between two sequences include GAP programs (Devereux, J., et al., Nucleic Acids Research 12 (1): 387 (1984)), BLASTP, BLASTN (Altschul, SF) in the GCG program package. et al., J. Molec. Biol. 215: 403-410 (1990)), and FASTA (Pearson and Lipman Proc. Natl. Acad. Sci. USA 85; 2444-2448 (1988)), but are not limited to these. It doesn't work. The BLAST group in the program is NCBI and other sources (BLAST Manual, Altschul, S., et al., NCBI NLM NIH Bethesda, MD 20894; Altschul, S., et al., J. Mol. Biol. 215: 403 -410 (1990)). The well known Smith Waterman algorithm can also be used to determine identity.
Parameters for comparing polypeptide sequences include the following:
Algorithm: Needleman and Bunsch [J. Mol Biol. 48: 443-453 (1970)]
Comparative Matrix: BLOSSUM62 (Henikoff and Henikoff, Proc. Natl. Acad. Sci. USA. 89: 10915-10919 (1992))
Gap Penalty: 8
Gap Length Penalty: 2
Useful programs in connection with these parameters are publicly available as the "gap" program of the Genetics Computer Group (Madison WI). The above mentioned parameters are the default parameters for peptide comparison (where there is no penalty for end gaps).
Parameters for polynucleotide comparisons include the following:
Algorithm: Needleman and Bunsh [J. Mol Biol. 48: 443-453 (1970)]
Comparison Matrix: Match = +10, Mismatch = 0
Gap Penalty: 50
Gap Length Penalty: 3
Available Programs: The "Gap" program of the Genetics Computer Group (Madison WI). These parameters are the default parameters for nucleic acid comparison.
The preferred meaning of "identity" for polynucleotides and polypeptides is optionally defined by the following (1) and (2).
(1) A polynucleotide embodiment comprises an isolated polynucleotide comprising a polynucleotide sequence having at least 50, 60, 70, 80, 85, 90, 95, 97 or 100% identity with a reference sequence of SEQ ID NO: 1. Further comprising, the polynucleotide sequence may be identical to the reference sequence of SEQ ID NO: 1 or may comprise a nucleotide change of less than a certain integer compared to the reference sequence, wherein the change is one or more nucleotide deletions, transitions selected from the group consisting of substitutions or insertions, including transitions and transversions, wherein the change is in a form interspersed separately between nucleotides of the reference sequence or in one or more consecutive groups within the reference sequence, May occur at the 5 'or 3' terminal position of the reference nucleotide sequence or at any position between these terminal positions. The number of nucleotide changes is determined by multiplying the total number of nucleotides of SEQ ID NO: 1 by an integer representing percent identity and dividing by 100 and subtracting this product from the total number of nucleotides of SEQ ID NO: 1. Or; Determined by the following formula:
_{n n ≤x n - (x n} · y)
Where n _n is the number of nucleotide changes, x _n is the total number of nucleotides of SEQ ID NO: 1, y is 0.50 for 50%, 0.60 for 60%, 0.70 for 70%, 0.70 for 80% 0.80, 0.85 for 85%, 0.90 for 90%, 0.95 for 95%, 0.97 for 97%, or 1.00 for 100%, · is a symbol for the multiplication operator, and any integer of x _n and y The other enemies are truncated to their nearest integer before subtracting from x _n . Changes in the polynucleotide sequence encoding the polypeptide of SEQ ID NO: 2 can generate nonsense, missense or frameshift mutations in this coding sequence, thereby changing the polypeptide encoded by the polynucleotide in which this change has occurred.
By way of example, a polypeptide sequence of the invention may be identical to the reference sequence of SEQ ID NO: 1 (ie, may be 100% identical), or may be a specific integer compared to the reference sequence such that the percent identity is less than 100% identity. The following nucleic acid changes may be included. Such changes are selected from the group consisting of one or more nucleic acid deletions, substitutions, or insertions, including transitions and translations, wherein the changes are interspersed separately between nucleotides of the reference sequence or interspersed in one or more consecutive groups within the reference sequence. In the form, it may occur at the 5 'or 3' terminal position of the reference polynucleotide sequence, or at any position between these terminal positions. The number of nucleic acid changes is determined by multiplying the total number of nucleic acids of SEQ ID NO: 1 by an integer representing percent identity and dividing by 100 and subtracting this product from the total number of nucleic acids of SEQ ID NO: 1; Determined by the following formula:
_{n n ≤x n - (x n} · y)
Wherein n _n is the number of nucleic acid changes, x _n is the total number of nucleic acids of SEQ ID NO: 1, y is, for example, 0.70 for 70%, 0.80 for 80%, 0.85 for 85%, etc. , · Is a symbol for the multiplication operator, and truncates the prime to the nearest integer before subtracting from x _n any number other than x _n and y.
(2) Polypeptide embodiments further comprise an isolated polypeptide comprising a polypeptide having at least 50, 60, 70, 80, 85, 90, 95, 97 or 100% identity with the polypeptide reference sequence of SEQ ID NO: 2 Wherein the polypeptide sequence may be identical to the reference sequence of SEQ ID NO: 2 or may comprise an amino acid change of less than a certain integer as compared to the reference sequence, wherein the change is one or more amino acid deletions, conservative and non-conserved An amino-terminal position of a reference polypeptide sequence, wherein the change is selected from the group consisting of substitutions, insertions, including red substitutions, wherein the change is in the form of interspersed individually between nucleotides of the reference sequence or in one or more contiguous groups within the reference sequence Or may occur at the carboxy-terminal position or at any position between these terminal positions, and may be used for The number of SEQ ID NO: after dividing it by 100 multiplied by an integer representing the total number of amino acid identity and percent of 2, SEQ ID NO: determining naemeurosseo remove these less from the total number of amino acids, or 2; Determined by the following formula:
_{n a ≤x a - (x a} · y)
Where n _a is the number of amino acid changes, x _a is the total number of amino acids of SEQ ID NO: 2, y is 0.50 for 50%, 0.60 for 60%, 0.70 for 80%, 80% for 80% 0.80, 0.85 for 85%, 0.90 for 90%, 0.95 for 95%, 0.97 for 97% or 1.00 for 100%, · is a symbol for the multiplication operator, and any integer of x _a and y The other enemies are truncated to their nearest integer before subtracting from x _a .
By way of example, a polypeptide sequence of the present invention may be identical to the reference sequence of SEQ ID NO: 2 (ie, may be 100% identical), or a specific integer compared to the reference sequence such that the percent identity is less than 100% identity. The following amino acid changes may be included. Such changes are selected from the group consisting of one or more amino acid deletions, substitutions including conservative substitutions and non-conservative substitutions, or insertions, wherein the changes are interspersed separately between amino acids of the reference sequence or one or more consecutive In forms interspersed with groups, they may occur at the amino- or carboxy-terminal positions of the reference polypeptide sequence or at any position between these terminal positions. The number of amino acid changes for a given% identity is determined by multiplying the total number of amino acids of SEQ ID NO: 2 by an integer representing percent identity and dividing by 100 and subtracting this product from the total number of amino acids of SEQ ID NO: 2; It can be determined by the following formula.
_{n a ≤x a - (x a} · y)
Wherein n _a is the number of amino acid changes, x _a is the total number of amino acids of SEQ ID NO: 2, y is, for example, 0.70 for 70%, 0.80 for 80%, 0.85 for 85%, etc. Is a symbol for the multiplication operator, and truncates the prime to be the nearest integer before subtracting from x _a any number other than x _a and y.
"Object (s)", as used herein in connection with an organism, includes multicellular eukaryotes, including, but not limited to, welfare animals, mammals, ovids, bovine animals, apes, primates, and humans. it means.
By “isolated” is meant a state that has been changed “by human” from its natural state, ie, when it occurs naturally, it is changed, separated from its original environment, or both. For example, a polynucleotide or polypeptide originally present in a living organism is not in an "isolated" state, but the same polynucleotide or polypeptide isolated from a coexistent substance in its natural state is in an "isolated" state as used herein. Moreover, polynucleotides or polypeptides introduced into an organism by transformation, genetic engineering or any other recombinant method are "isolated" even if they still exist in the organism, which may or may not be alive.
“Polynucleotide (s)” generally means any polyribonucleotide or polydeoxyribonucleotide, and may be unmodified RNA or DNA, including a single stranded region and a double stranded region, or may be modified RNA or DNA Can be.
A “variant” means a polynucleotide or polypeptide that differs from a reference polynucleotide or polypeptide but retains essential properties. Typical variants of polynucleotides differ from another reference polynucleotide and nucleotide sequence. Changing the nucleotide of the variant may or may not change the amino acid sequence of the polypeptide encoded by the reference polynucleotide. Nucleotide changes can cause amino acid substitutions, additions, deletions, fusions and truncations of polypeptides encoded by reference sequences, as discussed below. Typical variants of the polypeptide differ in amino acid sequence from another reference polypeptide. In general, differences are limited such that the sequences of the reference polypeptide and the variant are generally very similar and identical in many regions. Variants and reference polypeptides may differ in amino acids by one or more substitutions, additions, deletions, optionally combined. The substituted or inserted amino acid residue may or may not be a residue encoded by the genetic code. Variants of polynucleotides or polypeptides may be naturally occurring variants, such as allelic variants, or variants not known to occur naturally. Non-naturally occurring variants of polynucleotides and polypeptides can be prepared by mutagenesis techniques or by direct synthesis.
“Disease (s)” includes, for example, otitis media in infants and children, elderly pneumonia, sinusitis, pathogenic infections and invasive diseases, chronic otitis media with hearing loss, fluid accumulation in the middle ear, auditory nerve damage, By any disease caused by or associated with infection by a bacterium, including delayed language acquisition, upper respiratory tract infection and inflammation of the middle ear.
The following examples are well known to those skilled in the art, except as described in detail, and are performed using standard standard techniques. These examples are intended to illustrate the invention, not to limit the invention.
Example 1:
DNA sequencing for the discovery and identification of BASB027 gene from Moraxella catarrhalis strain ATCC 43617
The BASB027 gene of SEQ ID NO: 1 was first found in an Incyte PathoSeq database containing the incomplete genomic DNA sequence of Moraxella catarrhalis strain ATCC 43617 (also referred to as strain Mc2931). Translation of the BASB027 polynucleotide sequence set forth in SEQ ID NO: 2 showed significant similarity (32% identity at 817 amino acid overlaps) with the OMP85 outer membrane protein of Naaceria meningitidis.
The sequence of the BASB027 gene was further confirmed experimentally. To this end, genomic DNA is extracted from 10 ¹⁰ cells of Moraxella catarrhalis cells (strain ATCC 43617) using the QIAGEN Genomic DNA Extraction Kit (Qiagen Gmbh) and 1 μg of this material is primer E515515 (5′-ACT- ATA-GGG-CAC-GCG-TG-3 ') [SEQ ID NO: 5] and E515528: (5'-CCT-GCG-TTT-GTT-TGA-TTG-AG-3') [SEQ ID NO: 6 ] To amplify DNA by polymerase chain reaction. This PCR product was purified on a Biorobot 9600 (Qiagen Gmbh) apparatus and DNA was sequenced using the Big Dye Cycle Sequencing Kit (Perkin-Elmer) and ABI 377 / PRISM DNA Sequencer. . DNA sequencing was performed on both strands with an excess of 2 and full length sequences were assembled using the SeqMan program from the DNASTAR Lasergene software package. The resulting DNA sequence and putative polypeptide sequence were found as SEQ ID NO: 3 and SEQ ID NO: 4, respectively. The four nucleotide differences distinguish SEQ ID NO: 3 and SEQ ID NO: 4. Using the MEGALIGN program from the DNASTAR laserjet software package, alignment of the polynucleotide sequences of SEQ ID NO: 1 and 3: is shown in FIG. 2: Their level of identity was calculated to be 99.8%.
Using the same program, alignment of the polypeptide sequences of SEQ ID NOs: 2 and 4 was performed and is shown in Figure 3: Their level of identity was calculated to be 99.8%.
Example 2:
Mutation Analysis of BASB027 Gene in Several Moraxella Catalis Strains.
2A: Restriction Fragment Length Analysis (RFLP)
Genomic DNA was extracted from 16 Moraxella catalis (given in Table 1) as described below. Moraxella catalis was streaked to form single colonies on BHI agar plates and grown overnight at 37 ° C. Three or four single colonies were screened and used to inoculate about 1.5 ml Brain-heart infusion (BHI) broth seed culture, which was grown overnight at 37 ° C. in a 300 rpm shake incubator. Seed cultures were inoculated into 500 ml Erlenmeyer flasks containing about 150 ml of BHI broth and grown in a 175 rpm shake incubator at 37 ° C. for about 12-16 hours to generate cell masses for DNA isolation. Cells were collected by centrifugation with Sorvall GSA rotor at about 2000 X g for 15 minutes at room temperature. The supernatant was removed and the cell pellet suspended in about 5.0 ml of sterile water. Equivalent amount of lysis buffer (200 mM NaCl, 20 mM EDTA, 40 mM Tris-Hcl, pH 8.0, 0.5% (w / v) SDS, 0.5% (v / v) 2-mercaptoethanol and 250 μg / ml proteinase K) ) Was added and the cells were suspended by proper stirring and grinding. Cell suspensions were then incubated at 50 ° C. for about 12 hours to lyse bacteria and release chromosomal DNA. The proteinaceous material was precipitated by adding 5.0 ml of saturated NaCl (about 6.0 M in sterile water) and centrifuging at about 5,500 X g in a Sorvall SS34 rotor at room temperature. Chromosomal DNA was precipitated from the clearing supernatant by adding two volumes of 100% ethanol. Aggregated DNA was collected and washed by gentle stirring in a small amount of 70% ethanol solution. Purified chromosomal DNA was suspended in sterile water and dissolved / treated overnight at 4 ° C. by gentle rocking. The concentration of lysed DNA was 1.0 O.D. The spectrophotometer was measured at 260 nm using an absorption coefficient of about 50 μg / ml units.
Subsequently, this material was transferred to MC-D15-BamF (5'-AAG GGC CCA ATT ACG CAG AGG GGA TCC ACA GGA CTA CAG CGA GTG ACC ATT GAA AGC TTA C-3 ') [SEQ ID NO: 7] and MC- Amplified by PCR using D15-SalRC (AAG GGC CCA ATT ACG CAG AGG GTC GAC TTA TTA AAA GAC ACT ACC AAT CTG GAA CTG TAC CGT ATC G-3 ') [SEQ ID NO: 8] oligonucleotide. The corresponding BASB027 gene amplicons are then hydrolyzed independently using restriction enzymes (AciI, HindIII, MaeIII, NlaIII, RsaI, Sau3AI) and the restriction products are described in Molecular Cloning, a Laboratory Manual, Second Edition, Eds: Sambrook, Fritsch & Maniatis, Cold Spring Harbor press 1989 ”] were used to isolate by agarose or polyacrylamide gel electrophoresis using standard molecular biology methods. A photograph of the resulting electrophoretic gel is shown in FIG. 1. For each strain, RFLP patterns corresponding to six restriction enzymes were scored and combined. Thereafter, groups of strains sharing the same combination of RFLP patterns were defined. Using this method, the strains tested in this study were classified into four genome groups (Group 1: Mc2906, Mc2908, Mc2912, Mc2926; Group 2: Mc2905, Mc2907, Mc2909, Mc2911, Mc2913, Mc2960, Mc2975). Group 3: Mc2910, Mc2912, Mc2956, Mc2969; Group 4: Mc2931). These data support that the Moraxella catalis population used in this study exhibits limited nucleotide sequence diversity for the BASB027 gene.
Table 1: Characteristics of Moraxella catalys strains used in this study
StrainAn isolated countrySource Mc2904United States of AmericaTympanocentesis Mc2905United States of AmericaA high performer Mc2906United States of AmericaA high performer Mc2907United States of AmericaA high performer Mc2908United States of AmericaAcute otitis media Mc2909United States of AmericaA high performer Mc2910United States of AmericaA high performer Mc2911United States of AmericaAcute otitis media Mc2912United States of AmericaAcute otitis media Mc2913United States of AmericaAcute otitis media Mc2926United States of AmericaA high performer Mc2931 / ATCC43617United States of AmericaStadium suction Mc2956FinlandMiddle ear fluid Mc2960FinlandMiddle ear fluid Mc2969NorwayNasopharynx (pharyngitis-rhinitis) Mc2975NorwayNasopharynx (Rhinitis)
Example 3 Preparation of Plasmids for Expressing Recombinant BASB027
A: Cloning of BASB027
BamHI and SalI restriction sites engineered into forward ([SEQ ID NO: 7]) and reverse complementary ([SEQ ID NO: 8]) amplification primers, respectively, were incorporated into the commercial E. coli expression plasmid pQE30 (QiaGen, ampicillin resistance). Directing cloning of about 2500 bp PCR product was allowed to allow the mature BASB027 protein to be expressed as a fusion protein containing a (His) 6 affinity chromatography tag at the N-terminus. The BASB027 PCR product was purified from the amplification reaction using a spin column based on silica gel (QiaGen) according to the manufacturer's instructions. To generate the BamHI and SalI ends required for cloning, purified PCR products were sequentially digested with BamHI and SalI restriction enzymes as recommended by the manufacturer (Life Technologies). After performing the first restriction digest, the PCR product was purified using a spin column as above to remove salts and eluted in sterile water prior to the second enzyme digestion. The digested DNA fragments were once again purified using a spin column based on silica gel prior to ligation by the pQE30 plasmid.
B: Preparation of Expression Vector
To prepare the expression plasmid pQE30 for ligation, it was similarly digested with both BamHI and SalI and then treated with phosphatase (CIP, pmole at approximately 0.02 units / 5 'end, Life Technologies) of the calf in accordance with the manufacturer's instructions. Self-association. The ligation reaction proceeded as planned using about 5 fold molar excess of digested fragments of the prepared vector. About 20 μl of standard ligation reaction (about 16 ° C., about 16 hours) using methods well known in the art was performed using T4 DNA ligase (about 2.0 units / reaction, Life Technologies). An aliquot of ligation (approximately 5 μl) was used to transform electro-competent M15 (pREP4) cells according to methods well known in the art. After standing for about 2-3 hours at 37 ° C. in about 1.0 ml of LB broth, transformed cells were plated on LB agar plates containing kanamycin (50 μg / ml) and ampicillin (100 μg / ml). . Both of these antibiotics were included in the selection medium so that all transformed cells carry the lacIa gene necessary for the inhibition of expression of IPTG-inducible expression of the protein in pQE30, the pREP4 plasmid (KnR), and the pQE30-BASB027 plasmid (ApR). Make sure they are carrying both. Plates were incubated overnight at 37 ° C. for about 16 hours. Each KnR / ApR colony was screened with sterile toothpicks and used to "patch" inoculate new 1.0 LB KnR / ApR broth cultures as well as new LB KnR / ApR plates. Both patch plates and broth cultures were incubated overnight at 37 ° C. in a standard incubator (plate) or shaker bath.
PCR analysis based on whole cells was used to verify that the transformants contained the BASB027 DNA insert. Here, about 1.0 ml of overnight cultured LB Kn / Ap broth was transferred to 1.5 ml of polypropylene tube and centrifuged in a Beckmann microcentrifuge (about 3 minutes, room temperature, about 12,000 X g). Cells were collected by. The cell pellet was suspended in about 200 μl of sterile water and about 50 μl final volume PCR reaction containing both BASB027 forward and reverse amplification primers was performed as planned using about 10 μg aliquots. The final concentration of the PCR reaction components was essentially the same as that specified in Example 2 except that about 5.0 units of Taq polymerase were used. The initial 95 ° C. denaturation step was increased to 3 minutes to ensure thermal destruction of bacterial cells and release of plasmid DNA. Transformed solubilized using ABI Model 9700 thermal cycler and 32 cycles, 3-stage thermal amplification profile (ie 45 seconds at 95 ° C .; 45 seconds at 55-58 ° C. and 1 minute at 72 ° C.) BASB027 PCR fragments were amplified from sieve samples. After thermal amplification, approximately 20 μl aliquots of the reaction were analyzed by agarose gel electrophoresis (0.8% agarose in Tris-Acetate-EDTA (TAE) buffer). After gel electrophoresis and ethidium bromide staining, the DNA fragments were visualized by UV illumination. DNA molecules of standard size (1 Kb of ladder, Life Technologies) were electrophoresed side by side with the test sample and used to assess the size of the PCR product. The transformant that produced the expected 2500 bp PCR product was identified as a strain containing the BASB027 expression construct. The expression plasmid containing the strain was then analyzed for inducible expression of recombinant BASB027.
C: Analysis of expression of PCR positive transformants
For each PCR positive transformant identified above, about 5 ml of LB broth containing kanamycin (50 μg / ml) and ampicillin (100 μg / ml) was inoculated with cells from the patch plate and shaken Growing overnight at 37 ° C. (about 250 rpm). An aliquot (about 1.0 ml) of seed culture grown overnight was inoculated into a 125 ml Erlenmeyer flask containing about 25 ml of LB Kn / Ap broth, until the turbidity reached about 0.5 at OD600. That is, they were grown at 37 ° C. with shaking (about 250 rpm) up to the middle exponential phase (usually about 1.5 to 2.0 hours). At this time, almost half of the culture solution (about 12.5ml) was transferred to a 125ml flask, and IPTG (1.0M stock, Sigma manufactured in sterile water) was added to obtain a final concentration of 1.0mM. Caused expression. Incubation of both IPTG derived and non-derived cultures was continued at 37 ° C. for an additional about 4 hours with shaking. Samples containing both IPTG derived and non-derived cultures (about 1.0 ml) were removed after the inoculation period and cells were collected by centrifugation in a microcentrifuge at room temperature for about 3 minutes. Individual cell pellets are suspended in about 50 μl of sterile water, then mixed with the same volume of 2 × Laemelli SDS-PAGE sample buffer containing 2-mercaptoethanol and in a boiling bath for about 3 minutes. The protein was denatured by positioning. Equal volumes (approximately 15 μl) of crude IPTG derived cell lysate and uninduced cell lysate were placed on a double 12% Tris / Glycine polyacrylamide gel (1 mm thick Mini-gel, Novex). Loaded into. Induced and uninduced lysate samples were electrophoresed with molecular weight markers (SeeBlue, Novex) prestained under conventional conditions using standard SDS / Tris / Glycine running buffer (BioRad). After electrophoresis, one gel was stained with commassie light blue R250 (BioRad) and then decolorized to visualize the novel BASB027 IPTG inducible protein (s). Using a BioRad Mini-Protean II blotting device and Tobin methanol (20%) transfer buffer, the second gel was subjected to PVDF membrane (pore size 0.45 micron, Novex) at 4 ° C. for about 2 hours. ) And electroblotting. Blocking of the membrane and antibody culture were performed according to methods well known in the art. The expression and identity of the BASB027 recombinant protein was confirmed using a monoclonal anti-RGS (HIS) 3 antibody followed by a second rabbit anti-mouse antibody conjugated to HRP (QiaGen). Anti-His antibody reactivity patterns were visualized using ABT insoluble substrates or using Hyperfilm with the Amersham ECL chemiluminescence system.
D: sequence confirmation
To further demonstrate that the expressed IPTG inducible recombinant BASB027 protein is in the correct open reading frame and is not a false molecule resulting from the cloning artifact (ie, frame shift), the DNA sequence of the cloned insert is Decided. DNA sequences for Moraxella catalys BASB027 genes were obtained from one strand using conventional asymmetric PCR cycle sequencing methods (ABI Prism Dye-Terminator Cycle Sequencing, Perkin-Elmer). Sequencing reactions were performed with undigested expression plasmid DNA (about 0.5 μg / rxn) as a template and suitable pQE30 vector specific and ORF specific sequencing primers (about 3.5 pmol / rxn). In addition to the template and sequencing primers, each sequencing reaction (approximately 20 μl) contains four different dNTPs (ie, A, G, C and T) and four corresponding ddNTPs (ie, ddA, ddG, ddC and ddT). ) Contains a terminator nucleotide; Each end group is conjugated to one of four fluorescent dyes: Joe, Tam, Rox or Fam. The single stranded sequencing extension product was terminated at any position along the template by incorporation of a dye labeled ddNTP terminator. Fluorescent dye labeled termination products were purified using a microcentrifuge size exclusion chromatography column (Princeton Genetics), dried in vacuo, for template resuspension buffer (Perkin-Elmer) or PAGE for capillary electrophoresis. Dissolved in deionized formamide, denatured at 95 ° C. for about 5 minutes, high resolution capillary electrophoresis (ABI 310 automated DNA sequencer, Perkin-Elmer) or high resolution PAGE (ABI 377 automated DNA sequencer) Was analyzed using as recommended by the manufacturer. DNA sequence data generated from individual reactions were collected and relative fluorescence peak intensities were automatically analyzed on a PowerMAC computer using ABI sequence analysis software (Perkin-Elmer). Individually analyzed DNA sequences were manually edited for accuracy and then combined into consensus single stranded sequence "strings" using autoassembler software (Perkin-Elmer). Sequencing was used to determine whether the expression plasmid contained the correct sequence in the correct open reading frame.
Example 4: Preparation of Recombinant BASB027
Bacterial strains
Purified cell masses of recombinant proteins were prepared using recombinant expression strains of E. coli M15 (pREP4) containing a plasmid (pQE30) encoding BASB027 from Moraxella catalis. Expression strains were cultured on LB agar plates containing 50 μg / ml of kanamycin (“Kn”) and 100 μg / ml of ampicillin (“Ap”) to maintain both the pREP4 lacIq control plasmid and the pQE30-BASB027 expression construct. For cryopreservation at −80 ° C., the strains were grown in LB broth containing the same concentration of antibiotics and then mixed with the same volume of LB broth containing 30% (w / v) glycerol.
badge
Fermentation medium consisting of 2 × YT broth (Difco) containing 50 μg / ml Kn and 100 μg / ml Ap was used for the preparation of recombinant protein. Antifoam was added 0.25 ml / L to the fermentor broth (Antifoam 204, Sigma). To induce the expression of BASB027 recombinant protein, IPTG (isopropyl β-D-thiogalactopyranoside) was added to the fermentor (1 mM, final concentration).
Fermentation
Inoculate with several colonies from 0.3 ml of frozen cultures rapidly thawed into 500 ml Erlenmeyer seed flasks containing 50 ml working volume, or from selective agar plate cultures and 37 ± 1 for about 12 hours on a platform shaking at 150 rpm. Incubated at ° C (Innova 2100, New Brunswick Scientific). This seed culture was then used to inoculate a 5-L working volume fermentor containing both 2X YT broth and antibiotics Kn and Ap. The fermentor (Bioflo 3000, New Brunswick Scientific) was operated at 37 ± 1 ° C., 0.2 to 0.4 VVM air sparging, 250 rpm in a Rushton impeller. pH was not adjusted in flask seed culture or fermenter. During the fermentation, the pH was 6.5 to 7.3 in the fermentor. IPTG (1.0 M stock, made with sterile water) was added to the fermentor when the culture reached the median exponent (about 0.7 O.D. 600 units). Cells were induced for 2-4 hours and then harvested by centrifugation using 28RS Heraeus (Sepatech) or RC5C Ultrafast instruments. Cell paste was stored at −20 ° C. until processing.
refine
Chemicals and Materials
Grades of imidazole, guanidine hydrochloride, tris (hydroxymethyl), and EDTA (ethylenediamine tetraacetic acid) biotech ideals were obtained from Ameresco Chemical, Solon, Ohio. Triton X-100 (t-octylphenoxypolyethoxy-ethanol), sodium phosphate, monobasic, and urea were reagent grade or higher and were obtained from Sigma Chemical Company, St. Louis, Missouri. Glacial acetic acid and hydrochloric acid were obtained from Mallincrodt Baker Inc., Phillipsburg, New Jersey. Methanol was obtained from Fisher Scientific, Fairlawn, New Jersey. Pefabloc SC (4- (2-aminoethyl) -benzenesulfonylfluoride), a complete protease inhibitor cocktail tablet, and PMSF (phenylmethyl-sulfonylfluoride) were obtained from Roche Diagnostics Corporation, Indianapolis, Indiana. Vestatin, pepstatin A, and E-64 protease inhibitors were obtained from Calbiochem, LaJolla, California. Dulbecco's phosphate buffered saline (1XPBS) was obtained from Quality Biological, Inc., Gaithersburg, Maryland. Dulbecco's phosphate buffered saline (10 XPBS) was obtained from BioWhittaker, Walkersville, Maryland. BSA-free penta-His antibodies were obtained from QiaGen (QiaGen, Valencia, California). Peroxidase-conjugated AffiniPure goat anti-mouse IgG was obtained from Jackson Immuno Research, West Grove, Penn. AEC single solution was obtained from Zymed, South San Fransisco, California. All other chemicals were above reagent grade.
Ni-NTA superflow resin was obtained from Qiagen. Premade tris-glycine 4-20% and 10-20% polyacrylamide gels, all running buffers and solutions, SeeBlue Pre-Stained Standard, MultiMark Multi-Colored Standard And PVDF transfer membranes were obtained from Novex, San Diego, California. SDS-PAGE silver staining kit was obtained from Daiich Pure Chemicals Company Limited, Tokyo, Japan. Coomassie stain solution was obtained from Bio-Rad Laboratories, Hercules, California. PF 0.2 μm syringe filters were obtained from Pall Gelman Sciences, Ann Arbor, Michigan. GD / X 25 mm disposable syringe filters were obtained from Whatman Inc., Clifton, New Jersey. Dialysis tubing 8,000 MWCO was obtained from BioDesign Inc. Od New York, Carmal New York. BCA protein assay reagent and Snake skin dialysis tubing 3,500 MWCO were obtained from Pierce Chemical Co., Rockford, Illinois.
Extraction protocol
The cell paste was thawed for 30 to 60 minutes at room temperature. 5-6 g of material was metered into a 50 ml disposable centrifuge tube. To this was added 5 ml / g guanidine hydrochloride (Gu-HCl) buffer (6M guanidine hydrochloride, 100 mM sodium phosphate, monobasic, 10 mM Tris and 0.05% Triton X-100, pH 8.0). The cell paste was resuspended at 3/4 power for 1 minute using the PRO300D Progenic Homogenizer. The extracted mixture was then left with gentle stirring at room temperature for 60-90 minutes. After 60-90 minutes, the extraction mixture was centrifuged at 15,800 x g for 15 minutes (Sorvall RC5C centrifugation, 11,500 rpm). The supernatant (S1) was decanted and stored for further purification. Pellets (P1) were stored for analysis.
Bonding with BASB027 Nickel-NTA Resin
3-4 ml of Ni-NTA resin was added to S1. Then it was left at room temperature with gentle stirring for 1 hour. After 1 hour, S1 / Ni-NTA was packed into an XK16 Pharmacia column. The column was then washed with 1M Gu-HCl buffer (1M guanidine hydrochloride, 100 mM sodium phosphate, monobasic, 10 mM Tris and 0.05% Triton X-100, pH 8.0). It was then washed with phosphate buffer (100 mM sodium phosphate, monobasic, 10 mM Tris and 0.05% Triton X-100, pH 6.3). The protein was then eluted from the column using 250 mM imidazole buffer (250 mM imidazole, 100 mM sodium phosphate, monobasic, 10 mM Tris and 0.05% Triton X-100, pH 5.9).
Final formulation
BASB027 was formulated by dialysis overnight with three exchanges of 0.1% Triton X-100 and 1 × PBS, pH 7.4 to remove residual Gu-HCl and imidazole. Purified protein was characterized and used to prepare antibodies as follows.
Biochemical Characterization
SDS-PAGE and Western Blot Analysis
Purified recombinant protein was dissolved on a 4-20% polyacrylamide gel and electrophoretically transferred to PVDF membrane for 1 hour at 100 V as described above. Thebaine et al., 1979, Proc. Natl. Acad. Sci. USA 76: 4350-4354. The PVDF membrane was then pretreated with 25 ml of Dulbecco's phosphate buffered saline containing 5% skim milk powder. All subsequent incubations were performed using this pretreatment buffer.
PVDF membranes were incubated for 1 hour at room temperature with 25 ml of whole area serum or rabbit anti-His immune serum 1: 500 dilution. The PVDF membrane was then washed twice with wash buffer (containing 20 mM Tris buffer, pH 7.5, 150 mM sodium chloride and 0.05% Tween-20). PVDF membranes were incubated for 30 minutes at room temperature with 25 ml of a 1: 5000 dilution of peroxidase labeled goat anti-rabbit IgG (Jackson ImmunoResearch Laboratories, West Grove, PA). PVDF membranes were then washed four times with wash buffer and run for 10 minutes each with 3-amino-9-ethylcarbazole and urea peroxide supplied by Zymed (San Fransisco, Calif.).
Results of SDS-PAGE (FIG. 4) show a protein of about 95 kDa that is reactive to anti-RGS (His) antibody by Western blot of SDS-PAGE (FIG. 5).
Protein Sequencing
Amino terminal amino acid sequencing of the purified protein was performed using clearly defined protocols on the Hewlett-Packard Model G1000A sequencer of the Model 1090 LC and the Hewlett-Packard Model 241 sequencer of the Model 1100 LC to confirm the preparation of the correct recombinant protein. It was.
Example 5: Preparation of Antiserum Against Recombinant BASB027
Multivalent antiserum induced against BASB027 protein was generated by vaccinating two rabbits with purified recombinant BASB027 protein. Each animal was immunized three times in total intramuscular (i.m.) at approximately 21 days apart with about 20 μg BASB027 protein per injection (starting with complete Freund's adjuvant and then using incomplete Freund's adjuvant). Blood was drawn from animals prior to initial immunization (prior to blood) and on days 35 and 57.
Anti-BASB027 protein titers were measured by ELISA using purified recombinant BASB027 protein (0.5 μg / well). Titer is defined as the highest dilution that is at least 0.1 when calculated by the following equation: Average OD of two test sample antiserum—Average OD of two test sample buffers.
Antisera was used as the first antibody to identify the protein in the western blot described in Example 4 above. Western blots showed the presence of anti-BASB027 antibodies in the sera of immunized animals.
Example 6 Immunological Characterization
Western blot analysis
Several strains of Moraxella catalina were grown in 5% CO ₂ at 35 ° C. for 48 hours on chocolate agar plates. Several colonies were used to inoculate 25 ml of Mueller Hinton broth in a 250 ml flask. Cultures were grown overnight and collected by centrifugation. 30 μg of cells were then suspended and lysed in 150 μl of PAGE sample buffer (360 mM Tris buffer, pH 8.8, containing 4% sodium dodecyl sulfate and 20% glycerol), and the suspension was conditioned at 100 ° C. for 5 minutes. Incubated. The lysed cells were separated on 4-20% polyacrylamide gel and the separated proteins were electrophoretically transferred to PVDF membrane at 100 V for 1 hour as described above (Thebaine et al. 1979, Proc. Natl. Acad. Sci. USA. 76: 4350-4354). The PVDF membrane was then pretreated with 25 ml of Dulbecco phosphate buffered saline containing 5% skim milk powder. All subsequent incubations were performed using the pretreated buffer.
PVDF membranes were incubated for 1 hour at room temperature with 25 ml of 1: 500 dilution of pre-immune serum or rabbit immune serum. The PVDF membrane was then washed twice with wash buffer (20 mM Tris buffer at pH 7.5 containing 150 sodium chloride and 0.05% Tween-20). PVDF membranes were incubated for 30 minutes at room temperature using 25 ml of a 1: 5000 dilution of peroxide-labeled goat anti-rabbit IgG (Jackson ImmunoResearch Laboratories, West Grove, Pa.). The PVDF membrane was then washed four times with wash buffer and developed for 10 minutes each using 3-amino-9-ethylcarbasol, and urea peroxide, obtained from Zymed (San Francisco, Calif.).
A protein of about 95 kDa (corresponding to BASB027 expected molecular weight) that was reactive with antiserum was detected in all Moraxella strains (FIG. 7).
Bacterial activity
Complement mediated cytotoxic activity of the anti-BASB027 antibody was investigated to determine the vaccine potential of the BASB027 polypeptide. Antiserum was prepared as above. Activity of pre-immune serum and anti-BASB027 antiserum in mediating complement lethality of Moraxella catalis, except using cells from Moraxella catalys strains or cultivars instead of Neisseria meningitis cells And Serum Bactericidal Test "(Zollinger et al. (Immune Responses to Neisseria meningitis, in Manual of Clinical Laboratory Immunology, 3rd ed., Pg 347-349)).
Bactericidal titers of rabbit antiserum (50% lethality of homologous strains) were less than 1: 8 (preimmune) and greater than 1: 128 (immune).
Example 7: Presence of Antibodies to BASB027 in Human Recovery Phase Serum
Western blot analysis of purified recombinant BASB027 was performed in the same manner as in Examples 4 and 6 above, except that a human serum pool from a child infected with Moraxella catalis was used as the first antibody preparation. . The results indicate that antisera from naturally infected individuals react with the purified recombinant protein.
Example 8 Production of BASB027 Peptides, Antisera and Their Reactivity
Two short amino acid BASB027 specific peptides having sequences of CYAKPLNKKQNDQTDT (SEQ ID NO: 9) and YLTARRGQQTTLGEVVC (SEQ ID NO: 10) were prepared in the laboratory using generally known methods. These peptides, coupled with KLH, were used to produce antibodies in 12 week old specific pathogen free New Zealand female rabbits. Rabbits were injected four times with 200 μg of peptide-KLH in the form of complete Freund's adjuvant (first injection) or incomplete Freund's adjuvant (second, third, and fourth injection) at intervals of about three weeks. Animals were bled prior to the first immunization and 1 month after the fourth immunization.
Anti-peptide midpoint titers were determined by ELISA using free peptides. The anti-peptide midpoint titers after one month of four immunizations were better than 15000. Western blots of purified recombinant BASB027 using an anti-peptide antibody as the first antibody were prepared according to Examples 4 and 6 above. The result is shown in FIG.
Deposited substance
A deposit containing the Moraxella catalysed Catlin strain was deposited in US Type Culture Collection (“ATCC”) as Accession No. 43617, dated June 21, 1997. The deposit is described as Branhamella catarrhalis (Frosh and Kolle) and is a lyophilized 1.5-2.9 constructed from Moraxella catalis isolates obtained from the aspirate of coal miners with chronic bronchitis. It was an kb sized insertion library The deposit is described in Antimicrob. Agents Chemother. 21: 506-508 (1982).
The Moraxella catalis strain deposit is referred to as "deposit strain" or "dna of deposited strain".
The deposited strain contains the full-length BASB027 gene.
The vector pMC-HLY3, consisting of Moraxella catalys DNA inserted in pQE30, was deposited in US Type Culture Collection (ATCC) as accession no. 207106 on February 12, 1999.
The sequence of polynucleotides contained in the deposited strains / clones, and the amino acid sequences of any polynucleotides encoded thereby, controls the conflict with any sequence described herein.
Deposits of deposited strains have been deposited in accordance with the requirements of the Budapest Treaty on International Approval of Microbial Deposits in Patent Procedures. The deposit cannot be canceled and is provided to the public without any limitation or condition after the patent application. The deposited strains are merely for the convenience of those skilled in the art and are subject to patent requirements such as 35 U.S.C. It is not the same as that required in section 112.
SEQUENCE LISTING
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atgcgtaatt catattttaa aggttttcag gtcagtgcaa tgacaatggc tgtcatgatg 60
gtaatgtcaa ctcatgcaca agcggcggat tttatggcaa atgacattac catcacagga 120
ctacagcgag tgaccattga aagcttacaa agcgtgctgc cgtttcgctt gggtcaagtg 180
gtgagcgaaa accagttggc tgatggtgtc aaagcacttt atgcaacagg caatttttca 240
gatgtgcaag tctatcatca agaagggcgt atcatctatc aggtaaccga aaggccgtta 300
atcgctgaga ttaattttga gggcaatcgc ttaattccaa aagaaggtct acaagaaggg 360
ctaaaaaatg ctggcttagc tgtgggtcaa ccactaaaac aagccacagt acagatgatc 420
gaaaccgagc ttaccaatca atatatatca caaggctatt ataataccga aattactgtc 480
aaacagacga tgcttgatgg taatcgtgtt aagcttgata tgacctttgc tgaaggtaaa 540
cctgcacggg tggttgatat taatatcatt ggcaatcagc attttagcga tgcagatttg 600
attgatgtgc ttgcgattaa ggataataaa atcaatccac tgtctaaagc tgaccgttat 660
actcaagaaa agctggtgac cagtttagag aatttgcgtg ctaaatatct caatgcaggg 720
tttgtgcgtt ttgagattaa agatgctaag cttaatatta atgaagataa aaaccgtatc 780
tttgttgaga tttcattgca tgaaggtgag caatatcgct ttggacagac acagtttttg 840
ggtaatttaa cttatactca agcagaactt gaggcactgc ttaaattcaa agcagaagaa 900
gggttttcac aagccatgct tgagcaaaca acaaacaata tcagtaccaa atttggtgac 960
gatggctatt attatgctca aatccgtcct gtaacacgca ttaatgatga aagtcgtacg 1020
gttgatgtgg aatattatat tgaccctgta caccctgtct atgtacgccg tattaatttt 1080
acaggtaact ttaagaccca agatgaagta ctccgtcgtg agatgcgaca acttgaaggt 1140
gcgttggcat ctaatcaaaa aatccagctg tctcgtgcac gcttgatgcg gactgggttt 1200
tttaaacatg ttaccgttga tactcgtcca gtacccaact cacctgatca ggttgatgta 1260
aattttgtgg ttgaagaaca accttcagga tcatcaacca tcgcagcagg ctactctcaa 1320
agtggtggtg taacttttca atttgatgtt tctcaaaata actttatggg tacaggtaag 1380
cacgtcaatg cttcgttttc tcgctctgag acccgtgagg tgtatagttt gggtatgacc 1440
aacccatact ttaccgtaaa tggcgtctcg caaagcttga gtggctacta tcgtaaaacc 1500
aagtatgata acaagaacat tagtaattat gtacttgatt cttatggtgg ctcattaagc 1560
tatggatatc caattgatga aaatcaacgc ataagctttg gtctgaatgc tgacaatacc 1620
aagcttcatg gcggtcgttt tatgggcatt agtaatgtca agcagctgat ggcagatggt 1680
ggcaaaattc aagtggataa taatggcatt cctgatttta agcatgatta cacaacctac 1740
aatgccattt tggggtggaa ttattcaagt ctagatcgcc ctgtatttcc aacccaaggc 1800
atgagtcatt ctgtagattt gacggttggt tttggtgata aaactcatca aaaagtggtt 1860
tatcaaggca atatctatcg cccatttatc aaaaaatcag tcttgcgtgg atacgccaag 1920
ttaggctatg gcaataattt accattttat gaaaatttct atgcaggcgg ctatggttcg 1980
gttcgtggct atgatcaatc ctctttgggt ccacgctcac aagcctattt gacagctcgt 2040
cgtggtcaac aaaccacact aggagaggtt gttggtggta atgctttggc aactttcggc 2100
agtgagctga ttttaccttt gccatttaaa ggtgattgga tagatcaggt gcgtccagtg 2160
atattcattg agggcggtca ggtttttgat acaacaggta tggataaaca aaccattgat 2220
ttaacccaat ttaaagaccc acaagcaaca gctgaacaaa atgcaaaagc agccaatcgc 2280
ccgctactaa cccaagataa acagttgcgt tatagtgctg gtgttggtgc aacttggtat 2340
acgcccattg gtcctttatc tattagctat gccaagccat tgaataaaaa acaaaatgat 2400
cagaccgata cggtacagtt ccagattggt agtgtctttt aa 2442
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Met Arg Asn Ser Tyr Phe Lys Gly Phe Gln Val Ser Ala Met Thr Met
1 5 10 15
Ala Val Met Met Val Met Ser Thr His Ala Gln Ala Ala Asp Phe Met
20 25 30
Ala Asn Asp Ile Thr Ile Thr Gly Leu Gln Arg Val Thr Ile Glu Ser
35 40 45
Leu Gln Ser Val Leu Pro Phe Arg Leu Gly Gln Val Val Ser Glu Asn
50 55 60
Gln Leu Ala Asp Gly Val Lys Ala Leu Tyr Ala Thr Gly Asn Phe Ser
65 70 75 80
Asp Val Gln Val Tyr His Gln Glu Gly Arg Ile Ile Tyr Gln Val Thr
85 90 95
Glu Arg Pro Leu Ile Ala Glu Ile Asn Phe Glu Gly Asn Arg Leu Ile
100 105 110
Pro Lys Glu Gly Leu Gln Glu Gly Leu Lys Asn Ala Gly Leu Ala Val
115 120 125
Gly Gln Pro Leu Lys Gln Ala Thr Val Gln Met Ile Glu Thr Glu Leu
130 135 140
Thr Asn Gln Tyr Ile Ser Gln Gly Tyr Tyr Asn Thr Glu Ile Thr Val
145 150 155 160
Lys Gln Thr Met Leu Asp Gly Asn Arg Val Lys Leu Asp Met Thr Phe
165 170 175
Ala Glu Gly Lys Pro Ala Arg Val Val Asp Ile Asn Ile Ile Gly Asn
180 185 190
Gln His Phe Ser Asp Ala Asp Leu Ile Asp Val Leu Ala Ile Lys Asp
195 200 205
Asn Lys Ile Asn Pro Leu Ser Lys Ala Asp Arg Tyr Thr Gln Glu Lys
210 215 220
Leu Val Thr Ser Leu Glu Asn Leu Arg Ala Lys Tyr Leu Asn Ala Gly
225 230 235 240
Phe Val Arg Phe Glu Ile Lys Asp Ala Lys Leu Asn Ile Asn Glu Asp
245 250 255
Lys Asn Arg Ile Phe Val Glu Ile Ser Leu His Glu Gly Glu Gln Tyr
260 265 270
Arg Phe Gly Gln Thr Gln Phe Leu Gly Asn Leu Thr Tyr Thr Gln Ala
275 280 285
Glu Leu Glu Ala Leu Leu Lys Phe Lys Ala Glu Glu Gly Phe Ser Gln
290 295 300
Ala Met Leu Glu Gln Thr Thr Asn Asn Ile Ser Thr Lys Phe Gly Asp
305 310 315 320
Asp Gly Tyr Tyr Tyr Ala Gln Ile Arg Pro Val Thr Arg Ile Asn Asp
325 330 335
Glu Ser Arg Thr Val Asp Val Glu Tyr Tyr Ile Asp Pro Val His Pro
340 345 350
Val Tyr Val Arg Arg Ile Asn Phe Thr Gly Asn Phe Lys Thr Gln Asp
355 360 365
Glu Val Leu Arg Arg Glu Met Arg Gln Leu Glu Gly Ala Leu Ala Ser
370 375 380
Asn Gln Lys Ile Gln Leu Ser Arg Ala Arg Leu Met Arg Thr Gly Phe
385 390 395 400
Phe Lys His Val Thr Val Asp Thr Arg Pro Val Pro Asn Ser Pro Asp
405 410 415
Gln Val Asp Val Asn Phe Val Val Glu Glu Gln Pro Ser Gly Ser Ser
420 425 430
Thr Ile Ala Ala Gly Tyr Ser Gln Ser Gly Gly Val Thr Phe Gln Phe
435 440 445
Asp Val Ser Gln Asn Asn Phe Met Gly Thr Gly Lys His Val Asn Ala
450 455 460
Ser Phe Ser Arg Ser Glu Thr Arg Glu Val Tyr Ser Leu Gly Met Thr
465 470 475 480
Asn Pro Tyr Phe Thr Val Asn Gly Val Ser Gln Ser Leu Ser Gly Tyr
485 490 495
Tyr Arg Lys Thr Lys Tyr Asp Asn Lys Asn Ile Ser Asn Tyr Val Leu
500 505 510
Asp Ser Tyr Gly Gly Ser Leu Ser Tyr Gly Tyr Pro Ile Asp Glu Asn
515 520 525
Gln Arg Ile Ser Phe Gly Leu Asn Ala Asp Asn Thr Lys Leu His Gly
530 535 540
Gly Arg Phe Met Gly Ile Ser Asn Val Lys Gln Leu Met Ala Asp Gly
545 550 555 560
Gly Lys Ile Gln Val Asp Asn Asn Gly Ile Pro Asp Phe Lys His Asp
565 570 575
Tyr Thr Thr Tyr Asn Ala Ile Leu Gly Trp Asn Tyr Ser Ser Leu Asp
580 585 590
Arg Pro Val Phe Pro Thr Gln Gly Met Ser His Ser Val Asp Leu Thr
595 600 605
Val Gly Phe Gly Asp Lys Thr His Gln Lys Val Val Tyr Gln Gly Asn
610 615 620
Ile Tyr Arg Pro Phe Ile Lys Lys Ser Val Leu Arg Gly Tyr Ala Lys
625 630 635 640
Leu Gly Tyr Gly Asn Asn Leu Pro Phe Tyr Glu Asn Phe Tyr Ala Gly
645 650 655
Gly Tyr Gly Ser Val Arg Gly Tyr Asp Gln Ser Ser Leu Gly Pro Arg
660 665 670
Ser Gln Ala Tyr Leu Thr Ala Arg Arg Gly Gln Gln Thr Thr Leu Gly
675 680 685
Glu Val Val Gly Gly Asn Ala Leu Ala Thr Phe Gly Ser Glu Leu Ile
690 695 700
Leu Pro Leu Pro Phe Lys Gly Asp Trp Ile Asp Gln Val Arg Pro Val
705 710 715 720
Ile Phe Ile Glu Gly Gly Gln Val Phe Asp Thr Thr Gly Met Asp Lys
725 730 735
Gln Thr Ile Asp Leu Thr Gln Phe Lys Asp Pro Gln Ala Thr Ala Glu
740 745 750
Gln Asn Ala Lys Ala Ala Asn Arg Pro Leu Leu Thr Gln Asp Lys Gln
755 760 765
Leu Arg Tyr Ser Ala Gly Val Gly Ala Thr Trp Tyr Thr Pro Ile Gly
770 775 780
Pro Leu Ser Ile Ser Tyr Ala Lys Pro Leu Asn Lys Lys Gln Asn Asp
785 790 795 800
Gln Thr Asp Thr Val Gln Phe Gln Ile Gly Ser Val Phe
805 810
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atgcgtaatt catattttaa aggttttcag gtcagtgcaa tgacaatggc tgtcatgatg 60
gtaatgtcaa ctcatgcaca agcggcggat tttatggcaa atgacattgc catcacagga 120
ctacagcgag tgaccattga aagcttacaa agcgtgctgc cgtttcgctt gggtcaagtg 180
gtgagcgaag cacagttggc tgatggtgtc aaagcacttt atgcaacagg caatttttca 240
gatgtgcaag tctatcatca agaagggcgt atcatctatc aggtaaccga aaggccgtta 300
atcgctgaga ttaattttga gggcaatcgc ttaattccaa aagaaggtct acaagaaggg 360
ctaaaaaatg ctggcttagc tgtgggtcaa ccactaaaac aagccacagt acagatgatc 420
gaaaccgagc ttaccaatca atatatatca caaggctatt ataataccga aattactgtc 480
aaacagacga tgcttgatgg taatcgtgtt aagcttgata tgacctttgc tgaaggtaaa 540
cctgcacggg tggttgatat taatatcatt ggcaatcagc attttagcga tgcagatttg 600
attgatgtgc ttgcgattaa ggataataaa atcaatccac tgtctaaagc tgaccgttat 660
actcaagaaa agctggtgac cagtttagag aatttgcgtg ctaaatatct caatgcaggg 720
tttgtgcgtt ttgagattaa agatgctaag cttaatatta atgaagataa aaaccgtatc 780
tttgttgaga tttcattgca tgaaggtgag caatatcgct ttggacagac acagtttttg 840
ggtaatttaa cttatactca agcagaactt gaggcactgc ttaaattcaa agcagaagaa 900
gggttttcac aagccatgct tgagcaaaca acaaacaata tcagtaccaa atttggtgac 960
gatggctatt attatgctca aatccgtcct gtaacacgca ttaatgatga aagtcgtacg 1020
gttgatgtgg aatattatat tgaccctgta caccctgtct atgtacgccg tattaatttt 1080
acaggtaact ttaagaccca agatgaagta ctccgtcgtg agatgcgaca acttgaaggt 1140
gcgttggcat ctaatcaaaa aatccagctg tctcgtgcac gcttgatgcg gactgggttt 1200
tttaaacatg ttaccgttga tactcgtcca gtacccaact cacctgatca ggttgatgta 1260
aattttgtgg ttgaagaaca accttcagga tcatcaacca tcgcagcagg ctactctcaa 1320
agtggtggtg taacttttca atttgatgtt tctcaaaata actttatggg tacaggtaag 1380
cacgtcaatg cttcgttttc tcgctctgag acccgtgagg tgtatagttt gggtatgacc 1440
aacccatact ttaccgtaaa tggcgtctcg caaagcttga gtggctacta tcgtaaaacc 1500
aagtatgata acaagaacat tagtaattat gtacttgatt cttatggtgg ctcattaagc 1560
tatggatatc caattgatga aaatcaacgc ataagctttg gtctgaatgc tgacaatacc 1620
aagcttcatg gcggtcgttt tatgggcatt agtaatgtca agcagctgat ggcagatggt 1680
ggcaaaattc aagtggataa taatggcatt cctgatttta agcatgatta cacaacctac 1740
aatgccattt tggggtggaa ttattcaagt ctagatcgcc ctgtatttcc aacccaaggc 1800
atgagtcatt ctgtagattt gacggttggt tttggtgata aaactcatca aaaagtggtt 1860
tatcaaggca atatctatcg cccatttatc aaaaaatcag tcttgcgtgg atacgccaag 1920
ttaggctatg gcaataattt accattttat gaaaatttct atgcaggcgg ctatggttcg 1980
gttcgtggct atgatcaatc ctctttgggt ccacgctcac aagcctattt gacagctcgt 2040
cgtggtcaac aaaccacact aggagaggtt gttggtggta atgctttggc aactttcggc 2100
agtgagctga ttttaccttt gccatttaaa ggtgattgga tagatcaggt gcgtccagtg 2160
atattcattg agggcggtca ggtttttgat acaacaggta tggataaaca aaccattgat 2220
ttaacccaat ttaaagaccc acaagcaaca gctgaacaaa atgcaaaagc agccaatcgc 2280
ccgctactaa cccaagataa acagttgcgt tatagtgctg gtgttggtgc aacttggtat 2340
acgcccattg gtcctttatc tattagctat gccaagccat tgaataaaaa acaaaatgat 2400
cagaccgata cggtacagtt ccagattggt agtgtctttt aa 2442
<210> 4
<211> 813
<212> PRT
<213> Moraxella catarrhalis
<400> 4
Met Arg Asn Ser Tyr Phe Lys Gly Phe Gln Val Ser Ala Met Thr Met
1 5 10 15
Ala Val Met Met Val Met Ser Thr His Ala Gln Ala Ala Asp Phe Met
20 25 30
Ala Asn Asp Ile Ala Ile Thr Gly Leu Gln Arg Val Thr Ile Glu Ser
35 40 45
Leu Gln Ser Val Leu Pro Phe Arg Leu Gly Gln Val Val Ser Glu Ala
50 55 60
Gln Leu Ala Asp Gly Val Lys Ala Leu Tyr Ala Thr Gly Asn Phe Ser
65 70 75 80
Asp Val Gln Val Tyr His Gln Glu Gly Arg Ile Ile Tyr Gln Val Thr
85 90 95
Glu Arg Pro Leu Ile Ala Glu Ile Asn Phe Glu Gly Asn Arg Leu Ile
100 105 110
Pro Lys Glu Gly Leu Gln Glu Gly Leu Lys Asn Ala Gly Leu Ala Val
115 120 125
Gly Gln Pro Leu Lys Gln Ala Thr Val Gln Met Ile Glu Thr Glu Leu
130 135 140
Thr Asn Gln Tyr Ile Ser Gln Gly Tyr Tyr Asn Thr Glu Ile Thr Val
145 150 155 160
Lys Gln Thr Met Leu Asp Gly Asn Arg Val Lys Leu Asp Met Thr Phe
165 170 175
Ala Glu Gly Lys Pro Ala Arg Val Val Asp Ile Asn Ile Ile Gly Asn
180 185 190
Gln His Phe Ser Asp Ala Asp Leu Ile Asp Val Leu Ala Ile Lys Asp
195 200 205
Asn Lys Ile Asn Pro Leu Ser Lys Ala Asp Arg Tyr Thr Gln Glu Lys
210 215 220
Leu Val Thr Ser Leu Glu Asn Leu Arg Ala Lys Tyr Leu Asn Ala Gly
225 230 235 240
Phe Val Arg Phe Glu Ile Lys Asp Ala Lys Leu Asn Ile Asn Glu Asp
245 250 255
Lys Asn Arg Ile Phe Val Glu Ile Ser Leu His Glu Gly Glu Gln Tyr
260 265 270
Arg Phe Gly Gln Thr Gln Phe Leu Gly Asn Leu Thr Tyr Thr Gln Ala
275 280 285
Glu Leu Glu Ala Leu Leu Lys Phe Lys Ala Glu Glu Gly Phe Ser Gln
290 295 300
Ala Met Leu Glu Gln Thr Thr Asn Asn Ile Ser Thr Lys Phe Gly Asp
305 310 315 320
Asp Gly Tyr Tyr Tyr Ala Gln Ile Arg Pro Val Thr Arg Ile Asn Asp
325 330 335
Glu Ser Arg Thr Val Asp Val Glu Tyr Tyr Ile Asp Pro Val His Pro
340 345 350
Val Tyr Val Arg Arg Ile Asn Phe Thr Gly Asn Phe Lys Thr Gln Asp
355 360 365
Glu Val Leu Arg Arg Glu Met Arg Gln Leu Glu Gly Ala Leu Ala Ser
370 375 380
Asn Gln Lys Ile Gln Leu Ser Arg Ala Arg Leu Met Arg Thr Gly Phe
385 390 395 400
Phe Lys His Val Thr Val Asp Thr Arg Pro Val Pro Asn Ser Pro Asp
405 410 415
Gln Val Asp Val Asn Phe Val Val Glu Glu Gln Pro Ser Gly Ser Ser
420 425 430
Thr Ile Ala Ala Gly Tyr Ser Gln Ser Gly Gly Val Thr Phe Gln Phe
435 440 445
Asp Val Ser Gln Asn Asn Phe Met Gly Thr Gly Lys His Val Asn Ala
450 455 460
Ser Phe Ser Arg Ser Glu Thr Arg Glu Val Tyr Ser Leu Gly Met Thr
465 470 475 480
Asn Pro Tyr Phe Thr Val Asn Gly Val Ser Gln Ser Leu Ser Gly Tyr
485 490 495
Tyr Arg Lys Thr Lys Tyr Asp Asn Lys Asn Ile Ser Asn Tyr Val Leu
500 505 510
Asp Ser Tyr Gly Gly Ser Leu Ser Tyr Gly Tyr Pro Ile Asp Glu Asn
515 520 525
Gln Arg Ile Ser Phe Gly Leu Asn Ala Asp Asn Thr Lys Leu His Gly
530 535 540
Gly Arg Phe Met Gly Ile Ser Asn Val Lys Gln Leu Met Ala Asp Gly
545 550 555 560
Gly Lys Ile Gln Val Asp Asn Asn Gly Ile Pro Asp Phe Lys His Asp
565 570 575
Tyr Thr Thr Tyr Asn Ala Ile Leu Gly Trp Asn Tyr Ser Ser Leu Asp
580 585 590
Arg Pro Val Phe Pro Thr Gln Gly Met Ser His Ser Val Asp Leu Thr
595 600 605
Val Gly Phe Gly Asp Lys Thr His Gln Lys Val Val Tyr Gln Gly Asn
610 615 620
Ile Tyr Arg Pro Phe Ile Lys Lys Ser Val Leu Arg Gly Tyr Ala Lys
625 630 635 640
Leu Gly Tyr Gly Asn Asn Leu Pro Phe Tyr Glu Asn Phe Tyr Ala Gly
645 650 655
Gly Tyr Gly Ser Val Arg Gly Tyr Asp Gln Ser Ser Leu Gly Pro Arg
660 665 670
Ser Gln Ala Tyr Leu Thr Ala Arg Arg Gly Gln Gln Thr Thr Leu Gly
675 680 685
Glu Val Val Gly Gly Asn Ala Leu Ala Thr Phe Gly Ser Glu Leu Ile
690 695 700
Leu Pro Leu Pro Phe Lys Gly Asp Trp Ile Asp Gln Val Arg Pro Val
705 710 715 720
Ile Phe Ile Glu Gly Gly Gln Val Phe Asp Thr Thr Gly Met Asp Lys
725 730 735
Gln Thr Ile Asp Leu Thr Gln Phe Lys Asp Pro Gln Ala Thr Ala Glu
740 745 750
Gln Asn Ala Lys Ala Ala Asn Arg Pro Leu Leu Thr Gln Asp Lys Gln
755 760 765
Leu Arg Tyr Ser Ala Gly Val Gly Ala Thr Trp Tyr Thr Pro Ile Gly
770 775 780
Pro Leu Ser Ile Ser Tyr Ala Lys Pro Leu Asn Lys Lys Gln Asn Asp
785 790 795 800
Gln Thr Asp Thr Val Gln Phe Gln Ile Gly Ser Val Phe
805 810
<210> 5
<211> 17
<212> DNA
<213> Artificial Sequence
<220>
<223> Primer
<400> 5
actatagggc acgcgtg 17
<210> 6
<211> 20
<212> DNA
<213> Artificial Sequence
<220>
<223> Primer
<400> 6
cctgcgtttg tttgattgag 20
<210> 7
<211> 61
<212> DNA
<213> Artificial Sequence
<220>
<223> Oligonucleotide
<400> 7
aagggcccaa ttacgcagag gggatccaca ggactacagc gagtgaccat tgaaagctta 60
c 61
<210> 8
<211> 67
<212> DNA
<213> Artificial Sequence
<220>
<223> Oligonucleotide
<400> 8
aagggcccaa ttacgcagag ggtcgactta ttaaaagaca ctaccaatct ggaactgtac 60
cgtatcg 67
<210> 9
<211> 16
<212> PRT
<213> Artificial Sequence
<220>
<223> Oligopeptide
<400> 9
Cys Tyr Ala Lys Pro Leu Asn Lys Lys Gln Asn Asp Gln Thr Asp Thr
1 5 10 15
<210> 10
<211> 17
<212> PRT
<213> Artificial Sequence
<220>
<223> Oligopeptide
<400> 10
Tyr Leu Thr Ala Arg Arg Gly Gln Gln Thr Thr Leu Gly Glu Val Val
1 5 10 15
Cys
SEQUENCE INFORMATION
BASB027 Polynucleotide and Polypeptide Sequences
SEQ ID NO: 1
Moraxella catarrhalis BASB027 polynucleotide sequence from strain ATCC 43617
ATGCGTAATTCATATTTTAAAGGTTTTCAGGTCAGTGCAATGACAATGGCTGTCATGATG
GTAATGTCAACTCATGCACAAGCGGCGGATTTTATGGCAAATGACATTACCATCACAGGA
CTACAGCGAGTGACCATTGAAAGCTTACAAAGCGTGCTGCCGTTTCGCTTGGGTCAAGTG
GTGAGCGAAAACCAGTTGGCTGATGGTGTCAAAGCACTTTATGCAACAGGCAATTTTTCA
GATGTGCAAGTCTATCATCAAGAAGGGCGTATCATCTATCAGGTAACCGAAAGGCCGTTA
ATCGCTGAGATTAATTTTGAGGGCAATCGCTTAATTCCAAAAGAAGGTCTACAAGAAGGG
CTAAAAAATGCTGGCTTAGCTGTGGGTCAACCACTAAAACAAGCCACAGTACAGATGATC
GAAACCGAGCTTACCAATCAATATATATCACAAGGCTATTATAATACCGAAATTACTGTC
AAACAGACGATGCTTGATGGTAATCGTGTTAAGCTTGATATGACCTTTGCTGAAGGTAAA
CCTGCACGGGTGGTTGATATTAATATCATTGGCAATCAGCATTTTAGCGATGCAGATTTG
ATTGATGTGCTTGCGATTAAGGATAATAAAATCAATCCACTGTCTAAAGCTGACCGTTAT
ACTCAAGAAAAGCTGGTGACCAGTTTAGAGAATTTGCGTGCTAAATATCTCAATGCAGGG
TTTGTGCGTTTTGAGATTAAAGATGCTAAGCTTAATATTAATGAAGATAAAAACCGTATC
TTTGTTGAGATTTCATTGCATGAAGGTGAGCAATATCGCTTTGGACAGACACAGTTTTTG
GGTAATTTAACTTATACTCAAGCAGAACTTGAGGCACTGCTTAAATTCAAAGCAGAAGAA
GGGTTTTCACAAGCCATGCTTGAGCAAACAACAAACAATATCAGTACCAAATTTGGTGAC
GATGGCTATTATTATGCTCAAATCCGTCCTGTAACACGCATTAATGATGAAAGTCGTACG
GTTGATGTGGAATATTATATTGACCCTGTACACCCTGTCTATGTACGCCGTATTAATTTT
ACAGGTAACTTTAAGACCCAAGATGAAGTACTCCGTCGTGAGATGCGACAACTTGAAGGT
GCGTTGGCATCTAATCAAAAAATCCAGCTGTCTCGTGCACGCTTGATGCGGACTGGGTTT
TTTAAACATGTTACCGTTGATACTCGTCCAGTACCCAACTCACCTGATCAGGTTGATGTA
AATTTTGTGGTTGAAGAACAACCTTCAGGATCATCAACCATCGCAGCAGGCTACTCTCAA
AGTGGTGGTGTAACTTTTCAATTTGATGTTTCTCAAAATAACTTTATGGGTACAGGTAAG
CACGTCAATGCTTCGTTTTCTCGCTCTGAGACCCGTGAGGTGTATAGTTTGGGTATGACC
AACCCATACTTTACCGTAAATGGCGTCTCGCAAAGCTTGAGTGGCTACTATCGTAAAACC
AAGTATGATAACAAGAACATTAGTAATTATGTACTTGATTCTTATGGTGGCTCATTAAGC
TATGGATATCCAATTGATGAAAATCAACGCATAAGCTTTGGTCTGAATGCTGACAATACC
AAGCTTCATGGCGGTCGTTTTATGGGCATTAGTAATGTCAAGCAGCTGATGGCAGATGGT
GGCAAAATTCAAGTGGATAATAATGGCATTCCTGATTTTAAGCATGATTACACAACCTAC
AATGCCATTTTGGGGTGGAATTATTCAAGTCTAGATCGCCCTGTATTTCCAACCCAAGGC
ATGAGTCATTCTGTAGATTTGACGGTTGGTTTTGGTGATAAAACTCATCAAAAAGTGGTT
TATCAAGGCAATATCTATCGCCCATTTATCAAAAAATCAGTCTTGCGTGGATACGCCAAG
TTAGGCTATGGCAATAATTTACCATTTTATGAAAATTTCTATGCAGGCGGCTATGGTTCG
GTTCGTGGCTATGATCAATCCTCTTTGGGTCCACGCTCACAAGCCTATTTGACAGCTCGT
CGTGGTCAACAAACCACACTAGGAGAGGTTGTTGGTGGTAATGCTTTGGCAACTTTCGGC
AGTGAGCTGATTTTACCTTTGCCATTTAAAGGTGATTGGATAGATCAGGTGCGTCCAGTG
ATATTCATTGAGGGCGGTCAGGTTTTTGATACAACAGGTATGGATAAACAAACCATTGAT
TTAACCCAATTTAAAGACCCACAAGCAACAGCTGAACAAAATGCAAAAGCAGCCAATCGC
CCGCTACTAACCCAAGATAAACAGTTGCGTTATAGTGCTGGTGTTGGTGCAACTTGGTAT
ACGCCCATTGGTCCTTTATCTATTAGCTATGCCAAGCCATTGAATAAAAAACAAAATGAT
CAGACCGATACGGTACAGTTCCAGATTGGTAGTGTCTTTTAA
SEQ ID NO: 2
Moraxella catarrhalis BASB027 polypeptide sequence deduced from the polynucleotide sequence of SEQ ID NO: 1
MRNSYFKGFQVSAMTMAVMMVMSTHAQAADFMANDITITGLQRVTIESLQSVLPFRLGQV
VSENQLADGVKALYATGNFSDVQVYHQEGRIIYQVTERPLIAEINFEGNRLIPKEGLQEG
LKNAGLAVGQPLKQATVQMIETELTNQYISQGYYNTEITVKQTMLDGNRVKLDMTFAEGK
PARVVDINIIGNQHFSDADLIDVLAIKDNKINPLSKADRYTQEKLVTSLENLRAKYLNAG
FVRFEIKDAKLNINEDKNRIFVEISLHEGEQYRFGQTQFLGNLTYTQAELEALLKFKAEE
GFSQAMLEQTTNNISTKFGDDGYYYAQIRPVTRINDESRTVDVEYYIDPVHPVYVRRINF
TGNFKTQDEVLRREMRQLEGALASNQKIQLSRARLMRTGFFKHVTVDTRPVPNSPDQVDV
NFVVEEQPSGSSTIAAGYSQSGGVTFQFDVSQNNFMGTGKHVNASFSRSETREVYSLGMT
NPYFTVNGVSQSLSGYYRKTKYDNKNISNYVLDSYGGSLSYGYPIDENQRISFGLNADNT
KLHGGRFMGISNVKQLMADGGKIQVDNNGIPDFKHDYTTYNAILGWNYSSLDRPVFPTQG
MSHSVDLTVGFGDKTHQKVVYQGNIYRPFIKKSVLRGYAKLGYGNNLPFYENFYAGGYGS
VRGYDQSSLGPRSQAYLTARRGQQTTLGEVVGGNALATFGSELILPLPFKGDWIDQVRPV
IFIEGGQVFDTTGMDKQTIDLTQFKDPQATAEQNAKAANRPLLTQDKQLRYSAGVGATWY
TPIGPLSISYAKPLNKKQNDQTDTVQFQIGSVF
SEQ ID NO: 3
Moraxella catarrhalis BASB027 polynucleotide sequence from strain ATCC 43617
ATGCGTAATTCATATTTTAAAGGTTTTCAGGTCAGTGCAATGACAATGGCTGTCATGATG
GTAATGTCAACTCATGCACAAGCGGCGGATTTTATGGCAAATGACATTACCATCACAGGA
CTACAGCGAGTGACCATTGAAAGCTTACAAAGCGTGCTGCCGTTTCGCTTGGGTCAAGTG
GTGAGCGAAAACCAGTTGGCTGATGGTGTCAAAGCACTTTATGCAACAGGCAATTTTTCA
GATGTGCAAGTCTATCATCAAGAAGGGCGTATCATCTATCAGGTAACCGAAAGGCCGTTA
ATCGCTGAGATTAATTTTGAGGGCAATCGCTTAATTCCAAAAGAAGGTCTACAAGAAGGG
CTAAAAAATGCTGGCTTAGCTGTGGGTCAACCACTAAAACAAGCCACAGTACAGATGATC
GAAACCGAGCTTACCAATCAATATATATCACAAGGCTATTATAATACCGAAATTACTGTC
AAACAGACGATGCTTGATGGTAATCGTGTTAAGCTTGATATGACCTTTGCTGAAGGTAAA
CCTGCACGGGTGGTTGATATTAATATCATTGGCAATCAGCATTTTAGCGATGCAGATTTG
ATTGATGTGCTTGCGATTAAGGATAATAAAATCAATCCACTGTCTAAAGCTGACCGTTAT
ACTCAAGAAAAGCTGGTGACCAGTTTAGAGAATTTGCGTGCTAAATATCTCAATGCAGGG
TTTGTGCGTTTTGAGATTAAAGATGCTAAGCTTAATATTAATGAAGATAAAAACCGTATC
TTTGTTGAGATTTCATTGCATGAAGGTGAGCAATATCGCTTTGGACAGACACAGTTTTTG
GGTAATTTAACTTATACTCAAGCAGAACTTGAGGCACTGCTTAAATTCAAAGCAGAAGAA
GGGTTTTCACAAGCCATGCTTGAGCAAACAACAAACAATATCAGTACCAAATTTGGTGAC
GATGGCTATTATTATGCTCAAATCCGTCCTGTAACACGCATTAATGATGAAAGTCGTACG
GTTGATGTGGAATATTATATTGACCCTGTACACCCTGTCTATGTACGCCGTATTAATTTT
ACAGGTAACTTTAAGACCCAAGATGAAGTACTCCGTCGTGAGATGCGACAACTTGAAGGT
GCGTTGGCATCTAATCAAAAAATCCAGCTGTCTCGTGCACGCTTGATGCGGACTGGGTTT
TTTAAACATGTTACCGTTGATACTCGTCCAGTACCCAACTCACCTGATCAGGTTGATGTA
AATTTTGTGGTTGAAGAACAACCTTCAGGATCATCAACCATCGCAGCAGGCTACTCTCAA
AGTGGTGGTGTAACTTTTCAATTTGATGTTTCTCAAAATAACTTTATGGGTACAGGTAAG
CACGTCAATGCTTCGTTTTCTCGCTCTGAGACCCGTGAGGTGTATAGTTTGGGTATGACC
AACCCATACTTTACCGTAAATGGCGTCTCGCAAAGCTTGAGTGGCTACTATCGTAAAACC
AAGTATGATAACAAGAACATTAGTAATTATGTACTTGATTCTTATGGTGGCTCATTAAGC
TATGGATATCCAATTGATGAAAATCAACGCATAAGCTTTGGTCTGAATGCTGACAATACC
AAGCTTCATGGCGGTCGTTTTATGGGCATTAGTAATGTCAAGCAGCTGATGGCAGATGGT
GGCAAAATTCAAGTGGATAATAATGGCATTCCTGATTTTAAGCATGATTACACAACCTAC
AATGCCATTTTGGGGTGGAATTATTCAAGTCTAGATCGCCCTGTATTTCCAACCCAAGGC
ATGAGTCATTCTGTAGATTTGACGGTTGGTTTTGGTGATAAAACTCATCAAAAAGTGGTT
TATCAAGGCAATATCTATCGCCCATTTATCAAAAAATCAGTCTTGCGTGGATACGCCAAG
TTAGGCTATGGCAATAATTTACCATTTTATGAAAATTTCTATGCAGGCGGCTATGGTTCG
GTTCGTGGCTATGATCAATCCTCTTTGGGTCCACGCTCACAAGCCTATTTGACAGCTCGT
CGTGGTCAACAAACCACACTAGGAGAGGTTGTTGGTGGTAATGCTTTGGCAACTTTCGGC
AGTGAGCTGATTTTACCTTTGCCATTTAAAGGTGATTGGATAGATCAGGTGCGTCCAGTG
ATATTCATTGAGGGCGGTCAGGTTTTTGATACAACAGGTATGGATAAACAAACCATTGAT
TTAACCCAATTTAAAGACCCACAAGCAACAGCTGAACAAAATGCAAAAGCAGCCAATCGC
CCGCTACTAACCCAAGATAAACAGTTGCGTTATAGTGCTGGTGTTGGTGCAACTTGGTAT
ACGCCCATTGGTCCTTTATCTATTAGCTATGCCAAGCCATTGAATAAAAAACAAAATGAT
CAGACCGATACGGTACAGTTCCAGATTGGTAGTGTCTTTTAA
SEQ ID NO: 4
Moraxella catarrhalis BASB027 polypeptide sequence deduced from the polynucleotide sequence of SEQ ID NO: 3
MRNSYFKGFQVSAMTMAVMMVMSTHAQAADFMANDITITGLQRVTIESLQSVLPFRLGQV
VSENQLADGVKALYATGNFSDVQVYHQEGRIIYQVTERPLIAEINFEGNRLIPKEGLQEG
LKNAGLAVGQPLKQATVQMIETELTNQYISQGYYNTEITVKQTMLDGNRVKLDMTFAEGK
PARVVDINIIGNQHFSDADLIDVLAIKDNKINPLSKADRYTQEKLVTSLENLRAKYLNAG
FVRFEIKDAKLNINEDKNRIFVEISLHEGEQYRFGQTQFLGNLTYTQAELEALLKFKAEE
GFSQAMLEQTTNNISTKFGDDGYYYAQIRPVTRINDESRTVDVEYYIDPVHPVYVRRINF
TGNFKTQDEVLRREMRQLEGALASNQKIQLSRARLMRTGFFKHVTVDTRPVPNSPDQVDV
NFVVEEQPSGSSTIAAGYSQSGGVTFQFDVSQNNFMGTGKHVNASFSRSETREVYSLGMT
NPYFTVNGVSQSLSGYYRKTKYDNKNISNYVLDSYGGSLSYGYPIDENQRISFGLNADNT
KLHGGRFMGISNVKQLMADGGKIQVDNNGIPDFKHDYTTYNAILGWNYSSLDRPVFPTQG
MSHSVDLTVGFGDKTHQKVVYQGNIYRPFIKKSVLRGYAKLGYGNNLPFYENFYAGGYGS
VRGYDQSSLGPRSQAYLTARRGQQTTLGEVVGGNALATFGSELILPLPFKGDWIDQVRPV
IFIEGGQVFDTTGMDKQTIDLTQFKDPQATAEQNAKAANRPLLTQDKQLRYSAGVGATWY
TPIGPLSISYAKPLNKKQNDQTDTVQFQIGSVF
SEQ ID NO: 5
ACT ATA GGG CAC GCG TG
SEQ ID NO: 6
CCT GCG TTT GTT TGA TTG AG
SEQ ID NO: 7
AAG GGC CCA ATT ACG CAG AGG GGA TCC ACA GGA CTA CAG CGA GTG ACC ATT GAA AGC TTA C
SEQ ID NO: 8
AAG GGC CCA ATT ACG CAG AGG GTC GAC TTA TTA AAA GAC ACT ACC AAT CTG GAA CTG TAC CGT ATC G
SEQ ID NO: 9
CYAKPLNKKQNDQTDT
SEQ ID NO: 10
YLTARRGQQTTLGEVVC

权利要求:
Claims (24)
[1" claim-type="Currently amended] An isolated polypeptide comprising an amino acid sequence having at least 85% identity to an amino acid sequence selected from the group consisting of SEQ ID NO: 2 and SEQ ID NO: 4.
[2" claim-type="Currently amended] The isolated polypeptide of claim 1, wherein the amino acid sequence has at least 95% identity to an amino acid sequence selected from the group consisting of SEQ ID NO: 2 and SEQ ID NO: 4.
[3" claim-type="Currently amended] The polypeptide of claim 1 comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 2 and SEQ ID NO: 4.
[4" claim-type="Currently amended] Isolated polypeptide of SEQ ID NO: 2 or SEQ ID NO: 4.
[5" claim-type="Currently amended] An immunogenic fragment of a polypeptide according to any one of claims 1 to 4, wherein the immunogenic activity of the immunogenic fragment is substantially the same as the polypeptide of SEQ ID NO: 2 or SEQ ID NO: 4.
[6" claim-type="Currently amended] An isolated polynucleotide comprising a nucleotide sequence encoding a polypeptide having at least 85% identity to the amino acid sequence of SEQ ID NO: 2 or 4, respectively, over the length of SEQ ID NO: 2 or 4; Or a nucleotide sequence complementary to such an isolated polynucleotide.
[7" claim-type="Currently amended] An isolated polynucleotide comprising a nucleotide sequence having at least 85% identity to the nucleotide sequence encoding the polypeptide of SEQ ID NO: 2 or 4 over the entire coding region; Or a nucleotide sequence complementary to such an isolated polynucleotide.
[8" claim-type="Currently amended] An isolated polynucleotide comprising a nucleotide sequence having at least 85% identity to the nucleotide sequence of SEQ ID NO: 1 or 3 over the entire length of SEQ ID NO: 1 or 3; Or a nucleotide sequence complementary to such an isolated polynucleotide.
[9" claim-type="Currently amended] The isolated polynucleotide of claim 6, having at least 95% identity to SEQ ID NO: 1 or 3. 10.
[10" claim-type="Currently amended] An isolated polynucleotide comprising a nucleotide sequence encoding a polypeptide of SEQ ID NO: 2 or SEQ ID NO: 4.
[11" claim-type="Currently amended] An isolated polynucleotide comprising a polynucleotide of SEQ ID NO: 1 or SEQ ID NO: 3.
[12" claim-type="Currently amended] SEQ ID NO: 2 or obtained by screening a suitable library using labeled probes having sequences of SEQ ID NO: 1 or SEQ ID NO: 3 or fragments thereof under stringent hybridization conditions An isolated polynucleotide comprising a nucleotide sequence encoding a polypeptide of SEQ ID NO: 4.
[13" claim-type="Currently amended] An expression vector or live recombinant microorganism comprising the isolated polynucleotide according to any one of claims 6 to 12.
[14" claim-type="Currently amended] A host cell comprising the expression vector of claim 13, which expresses an isolated polypeptide comprising an amino acid sequence having at least 85% identity to an amino acid sequence selected from the group consisting of SEQ ID NO: 2 and SEQ ID NO: 4 Subcellular fraction or membrane of a host cell.
[15" claim-type="Currently amended] A method of producing a polypeptide comprising an amino acid sequence having at least 85% identity to an amino acid sequence selected from the group consisting of SEQ ID NO: 2 and SEQ ID NO: 4, wherein the polypeptide of claim 14 is provided under conditions sufficient to produce such polypeptide. Culturing the host cell and recovering the polypeptide from the incubator.
[16" claim-type="Currently amended] A method of expressing a polynucleotide according to any one of claims 6 to 12, wherein the host cell is transformed with an expression vector comprising at least one of such polynucleotides and the expression of any one of these polynucleotides. Culturing the host cell under sufficient conditions.
[17" claim-type="Currently amended] A vaccine composition comprising an effective amount of a polypeptide according to any one of claims 1 to 5 and a pharmaceutically acceptable carrier.
[18" claim-type="Currently amended] A vaccine composition comprising an effective amount of a polynucleotide according to any one of claims 6 to 12 and a pharmaceutically effective carrier.
[19" claim-type="Currently amended] 19. The vaccine composition of claim 17 or 18, wherein the composition comprises at least one other Moraxella catarrhalis antigen.
[20" claim-type="Currently amended] An antibody that is immunospecific to a polypeptide or immunological fragment according to any one of claims 1 to 5.
[21" claim-type="Currently amended] Diagnosing Moraxella catarrhalis infection, including identifying a polypeptide according to any one of claims 1 to 5 or an antibody specific for said polypeptide present in a biological sample from an animal believed to be infected. How to.
[22" claim-type="Currently amended] Use of a composition comprising an immunologically effective amount of a polypeptide according to any one of claims 1 to 5 for the manufacture of a medicament for use in causing an immune response in an animal.
[23" claim-type="Currently amended] Use of a composition comprising an immunologically effective amount of a polynucleotide according to any one of claims 6 to 12 for the manufacture of a medicament for use in causing an immune response in an animal.
[24" claim-type="Currently amended] A therapeutic composition useful for treating a person suffering from Moraxella catarrhalis disease comprising at least one antibody directed against a polypeptide according to any one of claims 1 to 5 and a suitable pharmaceutical carrier.

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ES2274627T3|2007-05-16|

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IL139951D0|2002-02-10|

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NO327201B1|2009-05-11|

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DK1082435T3|2007-02-05|

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KR20060129108A|2006-12-14|

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NO20006112D0|2000-12-01|

引用文献:

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公开号 | 申请日 | 公开日 | 申请人 | 专利标题

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法律状态:
1998-06-03|Priority to GB9811945.6

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1998-06-03|Priority to GBGB9811945.6A

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1999-03-08|Priority to GBGB9905304.3A

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1999-03-08|Priority to GB9905304.3

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1999-05-31|Application filed by 장 스테판느, 스미스클라인 비이참 바이오로지칼즈 에스.에이.

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2001-06-25|Publication of KR20010052552A

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2008-05-16|Application granted

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2008-05-16|Publication of KR100830282B1

优先权:

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申请号 | 申请日 | 专利标题

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GB9811945.6|1998-06-03|

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GBGB9811945.6A|GB9811945D0|1998-06-03|1998-06-03|Novel compounds|

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GBGB9905304.3A|GB9905304D0|1999-03-08|1999-03-08|Novel compounds|

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GB9905304.3|1999-03-08|