• 专利附录
  • 专利说明
  • 权利要求
  • 类似技术
  • 同族专利
  • 引用文献
  • 法律状态
  • 优先权
    • 专利摘要:
    The present invention provides antimicrobial, antifungal and anticancer activity by using a gene of the extracellular membrane protein (pgsBCA) involved in polygamma glutamic acid synthesis derived from Bacillus sp. As a surface expression matrix capable of efficiently expressing foreign proteins on the surface of microorganisms. The present invention relates to a microorganism expressing the peptide antibiotics P5 and Anal3 having an amphiphilic structure expressed on the cell surface, a method of preparing them, and a use thereof.
    公开号:KR20040034780A
    申请号:KR1020020063379
    申请日:2002-10-17
    公开日:2004-04-29
    发明作者:성문희；홍승표；이종수；정창민；함경수；이동건；박윤경；김철중；부하령
    申请人:한국생명공학연구원；주식회사 바이오리더스；학교법인조선대학교；
    IPC主号:
    专利说明:

    Surface expression method of peptide antibiotics P5 and AN3 using polygamma glutamic acid synthetic gene derived from Bacillus sp. Strain and use thereof {Surface-expression method for peptides P5 and Anal3 using pgs A}
    [16] The present invention provides a method for expressing the peptide antibiotics P5 and Anal3 on the surface of a microorganism by using an extracellular membrane protein (pgsBCA) involved in the synthesis of polygamma glutamic acid from a Bacillus strain, and the peptide antibiotics P5 and Anal3 respectively. It relates to a method for producing a lactic acid strain expressed in and a use thereof.
    [17] Recently, the antibiotic resistance of bacteria caused by the abuse of antibiotics has caused a big problem. Indeed, the rate at which bacteria are resistant to new antibiotics occurs much faster than the rate at which analogues of new antibiotics are developed. As a precondition for bacteria to be resistant to antibiotics, they are resistant to antibiotics. Bacteria that are resistant to antibiotics stop growing in the presence of antibiotics at the usual concentration but do not die as a result. Resistance is due to the fact that when antibiotics inhibit cell wall synthase, the activity of bacterial autolytic enzymes, such as autolysin, does not occur, which is why penicillin activates endogenous hydrolytic enzymes. By killing bacteria, bacteria also inhibit their activity, resulting in survival during antibiotic treatment. Virtually all resistant bacteria are known to have resistance (Liu and Tomasz, J. Infect. Dis. , 152, 365-372, 1985). Development of novel antibiotics that can kill these antibiotic resistant bacteria There is a need and a need to provide pharmaceutical compositions for the effective treatment of such new antibiotics for the treatment of infection and inflammation of bacteria.
    [18] Thus, attempts to develop new antibiotics against bacteria have been attempted by many researchers. Among them, development of peptides showing antimicrobial activity is prominent. In nature, bacteria can synthesize peptides or small organic molecules to kill neighboring bacteria. These bacteriocins are structurally subdivided. The first is lantibiotics, the second is nonlantibiotics, and the third is secreted by signal peptides (Cintas et al.,J. Bad., 180, 1988-1994, 1998). Animals, including insects, also produce naturally occurring peptide antibiotics (Bevins et al.,Ann. Rev. Biochem., 59, 395-414, 1990). The peptide antibiotics are structurally divided into three groups. The first is a cysteine-rich sheet peptide, the second is a helical amphiphilic molecule, and the third is a proline-rich peptide (Mayasaki et. al.,Int. J. Antimicrob. Agents, 9, 269-280, 1998). These antimicrobial peptides are known to play an important role in host defense and the innate immune system (Boman, H. G.,Cell, 65: 205, 1991; Boman, H. G.,Annu. Rev. Microbiol., 13:61, 1995). These antimicrobial peptides have a variety of structures depending on the amino acid sequence, the most of these structures are free of cysteine residues such as cecropin, an antimicrobial peptide found in insects, and form an amphiphilic alpha helical. . There have been many studies on the antimicrobial activity of amphiphilic peptides, and the amphiphilic peptides reported so far are magainin 2 (MA), cecropin A (CA) and melittin (ME). Peptides and the like.
    [19] It has been known that conjugation peptides can be prepared by recombining some sequences of these peptides to produce new synthetic peptides with excellent antibacterial, antifungal or anticancer activity (Chan, HC, et al. FEBS Lett., 1989, 259). , 103; Wade, D., et al., Int. J. Pept. Res. 1992, 40, 429), the inventors have found that the affinity of the secropin A (CA) and magainin 2 (magainin 2) , MA) conjugated peptide (CA-MA) as a template to synthesize a new peptide antibiotic P5 confirmed the antimicrobial, antifungal and anticancer activity of P5 (Application No. 10-2001-0057837, Applicant School Chosun University) .
    [20] In addition, the amino terminal region of RPL1 derived from the Gram-negative anaerobic bacterium, Helicobacter pylori, has a perfect amphiphilic helix structure (Putsep, K. et al., Nature , 398, 671-). 672, 1999), since these amphiphilic peptides have a structure similar to the lipid component of cell membranes, the mechanism of action that destroys microorganisms by binding to cell membrane lipids of microorganisms or by altering them by affecting the potential of cell membranes. Is reported. Accordingly, the inventors designed and synthesized peptide derivatives with an affinity structure that increases the hydrophobic region by substituting a specific site sequence among the amino terminal sites of the RPL1 protein of Helicobacter pylori having an affinity with another amino acid, Synthetic peptides were prepared, Anal3 confirmed that the antimicrobial, antifungal and anticancer activity has revealed that the peptide derivative is an antibiotic peptide that can be used as antibiotics and anticancer agents (Application No. 10-2000-0078615, Applicant Ham Kyung-soo).
    [21] These new antibiotics exhibit antimicrobial activity by a different mechanism of action than conventional antibiotics, and thus have the advantage of less resistance. Therefore, peptide antibiotics have a high possibility of industrial application in the field of pharmaceutical food. However, the biggest obstacle to the industrial use of the above-mentioned peptide antibiotics is that they cannot provide these inexpensively and in large quantities by the methods so far. For example, the production of peptide antibiotics by chemical synthesis has a problem of low economic feasibility, and there is an attempt to produce peptide antibiotics by genetic engineering techniques using microorganisms, but the expression rate of peptide antibiotics is very low and purified. There is a problem that is difficult and the host expressing the peptide is killed by the expressed peptide. In order to solve this problem, there are attempts to express, purify and produce peptides using genes that neutralize the toxicity of the peptides in the host, but they also have purification and economic difficulties.
    [22] Accordingly, there is an urgent need for a method for mass-producing a peptide substance by a simpler method, and since the purification is simple or unnecessary, a method for industrially using the peptide substance is required.
    [23] The technique of attaching and expressing a desired protein on the cell surface of a microorganism is called cell surface display technology. Knowledge of the secretory mechanism of proteins is increased, and this is the technology applied by applying molecular biological knowledge to this knowledge. This cell surface expression technology expresses foreign proteins on the surface by using surface proteins of microorganisms such as bacteria and yeast as surface anchoring motifs.The production of recombinant live vaccines, peptide / antibody library production and screening, whole cell It is a technology that has various application ranges such as absorbent and whole cell bioconversion catalyst. In other words, the range of application of this technology depends on which protein is expressed on the cell surface, and thus the potential for industrial application using cell surface expression technology is considerable.
    [24] Surface expression mothers are the most important for successful cell surface expression techniques. The key to this technology is how to select and develop a matrix capable of expressing foreign proteins on the cell surface.
    [25] Therefore, the surface-expressing parent with the following properties should be selected. First, there should be a secretion signal that helps to pass through the intracellular membrane to send the foreign protein to the cell surface. Second, there should be a target signal that helps the foreign protein adhere to the surface of the extracellular membrane stably. Parents should be selected with conditions such that they are expressed in large quantities but have little effect on cell growth, and that they can be stably expressed regardless of the size of the fourth protein and without altering the three-dimensional structure of the foreign protein. However, the surface-expressing matrix that satisfies all of the above conditions has not been developed yet, and to date, the above-mentioned disadvantages are compensated for.
    [26] There are four major surface-expressing mothers known to be used so far, such as extracellular membrane proteins, lipid proteins, secretory proteins, and surface organ proteins such as flagella proteins. For Gram negative bacteria, LamB, PhoE [Charbit et al., J. Immunol., 139: 1658-1664 (1987); Agterberg et al., Vaccine, 8: 85-91 (1990)], and proteins present in the extracellular membrane, such as OmpA, were mainly used, and the lipoprotein TraT [Felici et al., J. Mol. Biol., 222: 301-310 (1991)], peptidoglycan associated lipoprotein (PAL) [Fuchs et al., Bio / Technology, 9: 1369-1372 (1991)] and Lpp [Francisco et al., Proc. Natl. Acad. Sci. USA, 489: 2713-2717 (1992), and the like, fimbriae proteins such as FimA or FimH adhesin of type 1 fimbriae [Hedegaard et al., Gene, 85: 115-124 (1989)], PapA pilu subunit The same pili protein was used as a cell surface expression matrix to try to express foreign protein. In addition, ice nucleation protein [Jung et al., Nat. Biotechnol, 16: 576-560 (1998), Jung et al., Enzyme Microb. Technol, 22 (5): 348-354 (1998), Lee et al., Nat. Biotechnol, 18: 645-648 (2000)], pullulanase of Klebsiela oxytoca [Kornacker et al., Mol. Microl., 4: 1101-1109 (1990)] and Neiseria 's IgA protease [Klauser et al., EMBO J., 9: 1991-1999 (1990)] have been reported to be used as surface expression matrix. In the case of Gram positive bacteria, there was a report that the protein A derived from Staphylococcus aureus was used as the surface expression matrix to express the malaria antigen effectively, and the surface coating protein of lactic acid bacteria was used for the surface display.
    [27] The present inventors have diligently studied and studied the use of polygamma glutamic acid synthetic gene (pgsBCA) derived from Bacillus sp. As a new surface expression matrix, and have effectively expressed foreign proteins on the surface of microorganisms using pgsBCA protein. A new vector and a method for expressing a large amount of foreign protein on the surface of a microorganism have been developed (Application No. 10-2001-48373 Applicant Bioleaders).
    [28] In the present invention, the present inventors have revealed that the extracellular membrane protein involved in the synthesis of polygammaglutamic acid has many advantages as a surface expression matrix for expressing foreign proteins on the cell surface due to its amino acid primary sequence structure and characteristics: First, poly Extracellular membrane proteins involved in the synthesis of gamma glutamic acid can be expressed in large amounts on the cell surface for the synthesis and secretion of polygamma glutamic acid. Second, the extracellular membrane proteins involved in the synthesis of expressed polygamma glutamic acid are cell cycles. It remains stable even in the resting phase. Third, it is structurally particularly protruded on the cell surface in the case of pgsA. Fourth, the extracellular membrane protein (pgsBCA), which is involved in the synthesis of polygammaglutamic acid, originates from Gram-positive bacteria. Not only is expressed in Gram-positive bacteria, but also And fifth, surface expression is possible even if only one or two or more of the genes pgsB, pgsC, and pgsA encoding the polygammaglutamic acid synthase complex are included in the vector for microbial surface expression.
    [29] An object of the present invention is to provide a means for mass production of peptide antibiotics simply and safely.
    [30] Specifically, the present invention provides antimicrobial, antifungal, and anticancer drugs by using the gene of the extracellular membrane protein (pgsBCA) involved in polygamma glutamic acid synthesis derived from Bacillus sp. As a surface-expressing matrix to efficiently express foreign proteins on the surface of microorganisms. It is an object of the present invention to provide a surface expression vector capable of expressing the peptide antibiotics P5 and Anal3 having an active affinity structure on the cell surface.
    [31] It is another object of the present invention to provide a method for surface expression of a peptide which enables a simple and safe mass production of the peptide antibiotic from a non-toxic microorganism transformed by the surface expression vector.
    [32] In another aspect, the present invention is to enable the use of the antimicrobial or antifungal material suspended solids obtained by the microbial surface of the microbial surface-expressing live microorganisms or peptide antibiotics prepared by the method described above The purpose.
    [1] BRIEF DESCRIPTION OF THE DRAWINGS The genetic map of the conversion vector pHCE1LB: pgsA-P5 for surface expression using the Gram-negative and positive microorganisms as a host.
    [2] Figures 2A and 2B are pictures showing the isolation of the surface expression conversion vector plasmid in the lactic acid bacteria transformed with the surface expression conversion vector (pHCE1LB: pgsA-P5) of the present invention and the protein expression pattern of the peptide antibiotic P5 fused with pgsA Western blotted with antibody.
    [3] 3A and 3B are plate photographs showing antifungal activity against trichosporon beigelli , the fungus of which lactic acid bacteria surface-expressing peptide antibiotic P5 in the present invention, and graphs showing the survival rate of Trichosporon beigelli .
    [4] 4A and 4B are plate photographs showing antifungal activity against lactic acid bacteria Lactobacillus Candida albicans surface-expressing peptide antibiotic P5 in the present invention and graphs showing the survival rate of Candida albicans .
    [5] Figure 5 is a photograph showing the antifungal activity of the antifungal activity against Lactobacillus Candida albicans (A), Aspergillus flavus (B), Trichosporon beigelli (C), the surface-expressing peptide antibiotic P5 in the present invention.
    [6] Figure 6 is a genetic map of the conversion vector pHCE1LB: pgsA-Anal3 for surface expression in the Gram-negative and positive microorganism host according to the present invention.
    [7] 7A and 7B are specific images of the surface expression conversion vector plasmid isolated from lactic acid bacteria transformed with the surface expression conversion vector (pHCE1LB: pgsA-Anal3) of the present invention and the protein expression pattern of the peptide antibiotic Anal3 fused with pgsA. Western blotted with antibody.
    [8] 8A and 8B are plate photographs showing antifungal activity against Candida albicans , a lactic acid bacterium that expresses the peptide antibiotic Anal3 in the present invention, and graphs showing the survival rate of Candida albicans .
    [9] 9 shows antifungal activity of lactic acid bacteria expressing the peptide antibiotic Anal3 in the present invention against Candida albicans (A), Aspergillus flavus (B), Trichosporon beigelli (C), and Trichophyton rubrum (D). Verified photo.
    [10] FIG. 10 shows Bifidobacterium longgum, Enterococcus faecalis, Lactococcus lactis, Lactobacillus ecido to measure the antimicrobial activity against other lactic acid bacteria of Lactobacillus surface-expressing peptide antibiotics P5 and Anal3 in the present invention. Photograph showing antimicrobial activity against Philus, Lactobacillus amyloborus and Streptococcus thermophilus.
    [11] FIG. 11 is a photograph confirming the presence of plasmid in Lactobacillus after heat-treating Lactobacillus surface-expressing peptide antibiotics P5 and Anal3 in the present invention, and a picture confirming the state of the antibiotic peptide P5 and Anal3 fused protein fused with pgsA.
    [12] Figure 12 is a genetic map of the surface expression vector, pGNBCA and the conversion vector pGNBCA-HB168, which is a Gram-negative microorganism as a host, in an indirect embodiment according to the present invention.
    [13] Figure 13 is an indirect embodiment according to the present invention, Western blotting picture and fluorescence-activated cells showing the surface expression of the surface antigen group protein of hepatitis B virus in Gram-negative microorganisms transformed with surface expression conversion vector pGNBCA-HB168 Screening results graph.
    [14] Figure 14 is a genetic map of the surface expression vector, pGNCA and conversion vector pGNCA-HB168 in an indirect embodiment according to the present invention.
    [15] 15 shows surface antigens of hepatitis B virus in Gram-negative microorganisms transformed with surface expression conversion vectors (pGNCA-HB168: A2, pGNA-HB168: A3 and pGNHB-A: A4) in an indirect embodiment according to the present invention. Western blotting photograph and fluorescence-activated cell screening measurement graph showing surface expression of GI proteins.
    [33] In order to achieve the above object, the present invention provides a microorganism surface expression vector comprising any one or two or more of the genes pgsB, pgsC and pgsA encoding a polygammaglutamic acid synthase complex, and a gene encoding a peptide antibiotic; It provides a strain transformed by the vector.
    [34] In particular, the present invention provides a vector for microbial surface expression and a strain transformed with the vector including pgsA among genes pgsB, pgsC and pgsA, and a gene encoding a peptide antibiotic.
    [35] In particular, the present invention provides a microorganism surface expression vector to which P5 and Anal3 are applied as a peptide antibiotic and a strain transformed by the vector. Specifically, the present invention includes pgsA in a polygamma glutamic acid synthetic gene derived from a Bacillus sp. Strain and connects the peptide antibiotic gene P5 or Anal3 to the C-terminus of pgsA to form P5 or Anal3 as a fusion protein. Surface expression conversion vectors pHCE1LB: pgsA-P5 and pHCE1LB: pgsA-Anal3, which can be expressed on the surface of Gram-negative and positive bacteria, and strains transformed by them are provided.
    [36] In addition, the present invention, in order to achieve the above object, culturing the strain transformed by the microorganism surface expression vector, and the antimicrobial suspended solids containing the microbial or peptide antibiotics surface-expressing peptide antibiotics, Provided are methods for use as antifungal or anticancer agents. Specifically, the present invention, the antimicrobial, antifungal and the like containing a transformed strain, or a bacterial suspension containing the expressed peptide or peptide antibiotic P5 and Anal3 on the surface using the microorganism surface expression vector and It provides a pharmaceutical composition for anticancer. That is, the present invention provides a use of a bacterial suspension containing microorganisms or peptide antibiotics expressing the peptide antibiotics P5 and Anal3 for antibacterial, antifungal and anticancer activity.
    [37] In the present invention, the genes pgsB, pgsC, and pgsA each consist of the nucleotide sequences set forth in SEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO: 3.
    [38] In the present invention, the pHCE1LB plasmid was used as the base vector to enable replication and selection in both Gram-negative bacteria and Gram-positive bacteria. The expression vector pHCE1LB has a high-expression HCE promoter at all times, followed by a cloning site with various restriction enzyme sites, and an origin and ampicillin antibiotic marker that allows replication in Gram-negative bacteria. PHCE1LB also has a marker of origin and chloramphenicol antibiotics derived from lactobacillus that allows replication in Gram-positive bacteria. The use of the expression vector is described in detail in Korean Patent Application No. 10-2001-48373 of the applicant of the present invention.
    [39] Lactobacillus surface-expressing peptide antibiotics by the present invention are pathogenic fungi, for example Candida albicans , Trichosporon beigelii and Saccharomyces cerevisiae and Tricot It exhibits a fairly strong antifungal activity against Trichophyton rubrum . In addition, the lactic acid bacteria surface-expressing the peptide antibiotic of the present invention does not exhibit an antimicrobial action against other lactic acid bacteria.
    [40] On the other hand, according to the conventional literature, since the peptide antibiotics P5 and Anal3 revealed excellent antibacterial, antifungal and anticancer activity, the peptide antibiotics cultured by the present invention or the peptide antibiotics purified therefrom. It can also be predicted to show good antibacterial, antifungal and anticancer activity: (P5: Chan, HC, et al .. FEBS Lett., 1989, 259, 103; Wade, D., et al., Int. J. Pept. Res. 1992, 40, 429; Korean Patent Application No. 10-2001-0057837, Applicant School Corporation Chosun University), (Anal3 literature: Korean Patent Application No. 10-2000-0078615, Ham Kyung-su).
    [41] Thus, microorganisms (eg, lactic acid bacteria) or surface purified peptide antibiotics of the present invention exhibit excellent antibacterial, antifungal and anticancer effects as well as no cytotoxicity and are therefore safe for human body. And it can be usefully used as an anticancer substance.
    [42] The present invention will be described in more detail with reference to the following examples and indirect examples. These examples are only for illustrating the present invention in more detail, it will be apparent to those skilled in the art that the scope of the present invention is not limited to these examples.
    [43] In particular, in the following examples, peptide antibiotics P5 and Anal3 were used as foreign peptides, but any peptides showing other specific activities (antibacterial, antiviral, antifungal, anti-inflammatory, antiallergic, etc.) may be used.
    [44] In addition, in the following examples, although the Gram-positive bacteria Lactobacillus was used, it will be apparent to those skilled in the art that any other Gram-positive bacteria or Gram-positive bacteria can be obtained by transformation by the method according to the present invention.
    [45] In addition, in the following examples, the gene pgsBCA of the extracellular membrane protein involved in polygamma glutamic acid synthesis was obtained from Bacillus subtilis var.chungkookjang (KCTC 0697BP), but the gene was used for all Bacillus producing polygamma glutamic acid. A vector prepared by obtaining pgsBCA from the genus strain or a transformed microorganism using the vector will also be included in the scope of the present invention. For example, the preparation of a vector or the surface expression of a peptide antibiotic using a pgsBCA gene from another strain having 80% or more homology with the nucleotide sequence of the pgsBCA gene present in Bacillus subtilis Cheonggukjang is also included in the scope of the present invention. will be.
    [46] In addition, in the following examples, a surface expression vector was prepared using only pgsA among genes pgsBCA, but as can be inferred from the indirect examples, it is also possible to prepare a surface expression vector using only all or part of the gene pgsBCA. It will be included in the scope of.
    [47] Example 1 Preparation of Conversion Vector pHCE1LB: A-P5 for Surface Expression and Surface Expression of Peptide P5 Fused with pgsA
    [48] (1) A conversion vector pHCE1LB: pgsA-P5 for surface expression of the peptide antibiotic P5 was prepared.
    [49] PgsA among the genes of the extracellular membrane protein (pgsBCA) involved in polygamma glutamic acid synthesis derived from the Bacillus sp. Strain (Bacillus subtilis var.chungkookjang, KCTC 0697BP), hosts gram-negative and gram-positive microorganisms Intermediate vector pHCE1LB: pgsA was prepared by inserting into the basic vector pHCE1LB described above. In order to introduce the gene encoding the peptide antibiotic P5 into the intermediate vector, oligonucleotides having the nucleotide sequence of gene sequence 4 and sequence 5 encoding peptide P5 were mixed and denatured for 5 minutes at 95 ° C. and 37 Annenaling for 1 hour at ℃ to obtain a 65 bp double helix base.
    [50] 5'- ga tcc aag tgg aag aaa ctg ctc aag aaa ccg ctg ctc aag aag ctg ctc aag aaa ctg ta-3 'sequence 4
    [51] 5'- aag cta cag ttt ctt gag cag ctt ctt gag cag ccgg ttt ctt gag cag ttt ctt cca ctt g-3'SEQ ID NO.5
    [52] Both ends of the double-stranded base sequence of which the sequence 4 and the sequence 5 are annenaled are configured such that restriction sites BamH I and Hind III recognition sites present in the surface expression vector pHCE1LB: pgsA exist. The annealing P5 gene was mixed with the surface expression vectors pHCE1LB: pgsA previously treated with restriction enzymes BamH I and Hind III, and linked to each other so that the P5 gene was translated to the C-terminal region of the extracellular membrane protein gene (pgsA). Combined to match (ie, ORF). The conversion vector pHCE1LB: pgsA-P5 thus prepared is shown in FIG. 1.
    [53] Escherichia coli was transformed with the prepared surface expression vector pHCE1LB: pgsA-P5, and the Escherichia coli was deposited at the Korea Biotechnology Research Institute Gene Bank (KCTC, 52 Ueun-dong, Yuseong-gu, Daejeon) with an accession number of KCTC 10350BP.
    [54] (2) The surface expression vector pHCE1LB: pgsA-P5 according to the present invention was transformed and cultured in Lactobacillus casei to confirm whether the peptide P5 fused with pgsA was surface-expressed.
    [55] The transformed Lactobacillus strains were grown in a 500 ml flask containing 200 ml of MRS medium containing 50 mg / L of antibiotic chloramphenicol, followed by confirmation of the presence of pHCE1LB: pgsA-P5 plasmid in Lactobacillus (FIG. 2A), fusion protein expression of peptide P5 fused with pgsA was examined (FIG. 2B). Bacterial expression of the peptide antibiotic P5 fused with the C-terminus of the gene pgsA involved in polygamma glutamic acid synthesis was confirmed by SDS-polyacrylamide gel electrophoresis and western blotting using an antibody against pgsA. It was.
    [56] Specifically, surface expression was induced by propagating Lactobacillus casei transformed with pHCE1LB: pgsA-P5 at MRS medium (Lactobacillus MRS, Becton Dickinson and Company Sparks, USA) at 37 ° C. Expression-induced Lactobacillus protein was obtained at the same cell concentration and then denatured to prepare a sample, which was analyzed by SDS-polyacrylamide gel electrophoresis, and the fractionated proteins were then PVDF membranes (polyvinylidene-difluoride membranes). , Bio-Rad). The PVDF membrane to which the proteins were transferred was immersed in blocking buffer (50 mM Tris hydrochloric acid, 5% skim milk, pH 8.0), stirred for 1 hour, blocked, and then blocked with a rabbit-derived polyclonal primary antibody against pgsA. Diluted 1000 times in the buffer solution and reacted for 12 hours. After completion of the reaction, the membrane was washed with a buffer solution, and the secondary antibody against the rabbit conjugated with biotin was diluted 1000-fold in blocking buffer and reacted for 4 hours. After completion of the reaction, the membrane was washed with a buffer solution and washed again by reacting the avidin-biotin reagent for 1 hour. The washed membrane was colored by adding H202 and DAB solution as a substrate and a color reagent, and confirmed specific binding between the specific antibody to pgsA and the fusion protein (FIG. 2B). In FIG. 2B, lane 1 is an untransformed host cell, Lactobacillus casei, and lane 2 is a transformed pHCE1LB: pgsA-P5 / Lactobacillus casei. As shown in the figure, the fusion protein band of about 44 KDa was confirmed by pHCE1LB: pgsA-P5 plasmid. Since pgsA is about 41.8 KDa and peptide P5 is about 2.2 KDa, it can be seen that the band representing 44 KDa is a fusion protein in which pgsA and peptide P5 are fused.
    [57] Example 2 Measurement of Antifungal Activity of Lactobacillus Surface-expressing Peptide Antibiotic P5
    [58] (1) In order to measure the antifungal activity of the Lactobacillus surface-expressing peptide antibiotic P5 identified in Example 1, first target the pathogenic fungi Candida albicans (TIMM 1768) and tricosporone Beiglai (KCTC 7707) Visualization experiment of antifungal activity was performed.
    [59] Specifically, 50 μl of PDB medium (20% potato infusion frum, 2% bactodextrose) containing 2 × 10 3 various fungi was dispensed into a 96-well plate and containing Lactobacillus expressing P5 of the present invention. Dilutions were diluted 1/2 fold from 50 μl / well of MRS medium and added to the wells with fungi, followed by incubation at 37 ° C. for 16 hours, and the cultures were plated on PDB agar plates to visualize the strains. As a result, a large number of colonies could be found in the tricosporon Beiglai strain and the Candida albicans strain itself, and in the plate in which wild-type Lactobacillus was added to these strains (FIGS. 3A and 4A), the peptide antibiotic P5 of the present invention. When Lactobacillus surface-expressing was added, it was confirmed that colonies were not completely inhibited by the growth of bacteria, and the percentage of viable bacteria of these fungi was graphically represented (FIGS. 3B and 4B).
    [60] As can be seen from the above results, it was confirmed that Lactobacillus surface-expressing the peptide antibiotic P5 of the present invention shows excellent antifungal activity.
    [61] (2) In addition, the antifungal activity of Lactobacillus surface-expressing peptide antibiotic P5 of the present invention was confirmed by the pathogenic fungi Candida albicans (TIMM 1768), Tricosporon Beiglai (KCTC 7707) and Aspergillus flavers. The subject was identified through a scanning electron microscopy (SEM).
    [62] Specifically, Lactobacillus surface expression of wild-type Lactobacillus and P5 by culturing Candida albicans (TIMM 1768), Tricosporon Beiglai (KCTC 7707) and Saccharomyces cerbizia (KCTC 7296) by the above method After incubating the Bacillus at 37 ° C for 16 hours together, 0.2M sodium phosphate buffer solution in which 5% glutaraldehyde was dissolved in a certain amount of mixed culture medium was added to the same volume as the mixed culture solution for 2 hours at 4 ° C. Prepare the sample by fixing it for a while. The sample was filtered with an isopore filter (Isopore filters, 0.2m pore size, Millipore, Bedford. MA. USA) and washed with 0.1M sodium cacodylate buffer (Na-cacodylate buffer, pH 7.4), The filter was treated with 1% osmium tertroxide, washed with 5% sucrose (5% sucrose) dissolved in sodium cacodylate buffer, and dehydrated stepwise with ethanol. The sample thus treated was lyophilized, gold-coated and observed with a scanning electron microscope (HITACHI S-2400, Japan).
    [63] As a result, when Lactobacillus surface-expressing the peptide antibiotic P5 of the present invention was added to Candida albicans (FIG. 5A), Aspergillus plabus (FIG. 5B), and tricosporone Beiglai (FIG. 5C) It was confirmed that more cell destruction of fungi compared to the case (Fig. 5).
    [64] Example 3 Preparation of Conversion Vector pHCE1LB: A-Anal3 for Surface Expression and Surface Expression of Peptide Anal3 Fused with pgsA
    [65] (1) A conversion vector pHCE1LB: A-Anal3 capable of surface expressing the peptide antibiotic Anal3 was prepared by the same method as described in (1) of Example 1.
    [66] In order to introduce the gene encoding the peptide antibiotic Anal3 into the intermediate vector pHCE1LB: pgsA, the nucleotide sequences of the gene sequences 6 and 7 encoding the peptide Anal3 were annenaled in the same manner as in Example 1 to obtain a 62 bp double helix. The sequence was obtained.
    [67] 5'- ga tcc gcg aag aag gtg ttc aaa cgc ctg gag aag ctg ttt agc aaa atc tgg aac tgg aag ta-3'SEQ. 6
    [68] 5'- aag cta ctt cca gtt cca gat ttt gct aaa cag ctt ctc cag gcg ttt gaa cac ctt ctt cgc g-3'SEQ ID NO: 7
    [69] Both ends of the double-stranded nucleotide sequence formed by SEQ ID NO: 6 and SEQ ID NO: 7 was configured such that restriction sites BamH I and Hind III recognition sites present in the surface expression vector pHCE1LB: pgsA exist. The annealing Anal3 gene was previously linked to the C-terminal region of the extracellular membrane protein gene (pgsA) of the surface expression vector pHCE1LB: pgsA treated with BamH I and Hind III. The conversion vector pHCE1LB: pgsA-Anal3 thus prepared is shown in FIG. 6.
    [70] Escherichia coli was transformed with the surface expression vector, and Escherichia coli containing pHCE1LB: pgsA-Anal3 was deposited in the Korea Biotechnology Research Institute Gene Bank (KCTC, 52 Ueun-dong, Yuseong-gu, Daejeon) as an accession number of KCTC 10348BP.
    [71] (2) The present invention transformed the surface expression vector pHCE1LB: pgsA-Anal3 into Lactobacillus and confirmed the presence of pHCE1LB: pgsA-Anal3 plasmid in Lactobacillus (FIG. 7A), and the antibiotic peptide Anal3 fused with pgsA Expression of the fusion protein was investigated (FIG. 7B).
    [72] To this end, the expression vector was transformed into Lactobacillus and the expression was induced in the same manner as in Example 1, followed by SDS-expressing Lactobacillus of the antibiotic peptide Anal3 fused with the C-terminus of the gene pgsA, which synthesizes polygamma glutamic acid. Polyacrylamide gel electrophoresis and western blotting (western immunoblotting) with specific antibodies to pgsA were confirmed (FIG. 7B). In FIG. 2B, lane 1 is transformed pHCE1LB: pgsA-Anal3 / Lactobacillus case, and lane 2 is untransformed Lactobacillus case. As shown in the figure, the fusion protein band of about 44 KDa was confirmed by pHCE1LB: pgsA-Anal3 plasmid. Since pgsA is about 41.8 KDa and peptide Anal3 is about 2.2 KDa, it can be seen that the band representing 44 KDa is a fusion protein in which pgsA and peptide P5 are fused.
    [73] Example 4: Determination of antifungal activity of Lactobacillus surface-expressing peptide antibiotic Anal3
    [74] (1) In order to measure the antifungal activity of the Lactobacillus surface-expressing the peptide antibiotic substance Anal3 identified in Example 3, first to visualize the antifungal activity of the pathogenic fungi Candida albicans (TIMM 1768), Example The same procedure as in 2 was carried out. As a result, a large number of colonies were found in the Candida albicans strain itself and the plate to which the wild type Lactobacillus was added to these strains (FIG. 8A), but the Lactobacillus surface-expressing antibiotic peptide Anal3 of the present invention was added. In this case, the growth of the fungi was completely suppressed, so it was confirmed that colonies were not seen, and the percentage of the viable bacteria of these fungi was confirmed by the graph (FIG. 8B).
    [75] From the above results, it was confirmed that Lactobacillus surface-expressing the peptide antibiotic Anal3 of the present invention shows excellent antifungal activity.
    [76] (2) In addition, the antifungal activity of Lactobacillus surface-expressing antibiotic peptide Anal3 of the present invention can be found in the pathogenic fungi Candida albicans (TIMM 1768), Aspergillus plavers and Tricosporon Beigella (KCTC 7707) and Tricot The python ruburum was identified by scanning electron microscopy (SEM) in the same process as in Example 2.
    [77] As a result, Lactobacillus surface-expressing the peptide antibiotic Anal3 of the present invention was Candida albicans (FIG. 9A), Aspergillus plabus (FIG. 9B), Tricosporone Beiglai (FIG. 9C) and Tricopython luburum ( When added to Figure 9D) it was confirmed that more cell destruction of the fungi compared to the other case (Fig. 9).
    [78] Example 5 Measurement of Antimicrobial Activity against Lactic Acid Bacteria of Lactobacillus Surface-Expressing Peptide Antibiotics P5 and Anal3
    [79] Bifidobacterium longum, Enterococcus faecalis, Lactobacillus for measuring the antimicrobial activity against other lactic acid bacteria of Lactobacillus surface-expressing peptide antibiotics P5 and Anal3 identified in the above examples Visualization experiments were conducted on Lactococcus lactis, Lactobacillus acidophilus, Lactobacillus amylovorus, and Streptococcus thermophilus with antibacterial paper disc method. Was performed.
    [80] Specifically, each of the strains incubated in MRS medium was plated on an MRS agar plate by 1 x 10 3 , and diluted 1/2 of each Lactobacillus casei expressing the peptide antibiotics P5 and Anal3 by paper disc. After soaking in the above paper disc (ADVANTEC, Toyo Roshi Kaisha, Japan) was smeared on the lactic acid bacteria plated MRS agar plate was incubated for 16 hours at 37 ℃ and observed the ring formed around the paper disc.
    [81] As a result, Bifidobacterium longgum (A), Enterococcus pecalis (B), Lactococcus lactis (C), Lactobacillus ecidophilus (D), and Lactobacillus amyloborus as shown in FIG. E) and Streptococcus thermophilus (F) did not exhibit any antimicrobial activity against Lactobacillus casei expressing peptide antibiotics P5 and Anal3. In Figure 10, 1 is an untransformed Lactobacillus casei, 2, 3 is a transformed pHCE1LB: pgsA-P5 / lactobacillus casei, lanes 4, 5 are transformed pHCE1LB: pgsA-Anal3 / Lactobacillus casei to be.
    [82] That is, Lactobacillus casei expressing the peptide antibiotics P5 and Anal3 can be used in combination with lactic acid bacteria having different effects.
    [83] Example 6: Experiment to prevent side effects generated during genetic manipulation by heat treatment of expressed peptide antibiotic and plasmid
    [84] As the development and use of genetically modified foods (GMO) has increased, voices are concerned about the incidence of various diseases, such as the leakage of engineered genes (DNA) to other microorganisms or plants and animals, and cancer.
    [85] Therefore, in the present embodiment, when heat-treated the transformed microorganism according to the present invention, while maintaining the function as an antibiotic, while genetically engineered DNA (that is, plasmid) is damaged to solve the problem of GMO It was.
    [86] (1) First, it was confirmed whether the respective peptide antibiotics were maintained without being degraded even after heat treatment.
    [87] In the above-described embodiment, the lactobacillus transformed with the surface expression vectors pHCE1LB: pgsA-P5 and pHCE1LB: pgsA-Anal3, respectively, expresses the fusion protein of Anal3 fused with antibiotic peptides P5 and pgsA fused with pgsA, respectively. Confirmed.
    [88] Peptide fused with pgsA in lactobacillus after heat-treating Lactobacillus confirmed the expression of the fusion protein by Western blotting according to the same method as in Example 1 at 110 ° C. for 20 minutes. In order to determine the state of the fusion protein of antibiotics P5 and pgsA and Anal3, SDS-polyacrylamide gel electrophoresis and Western blotting using specific antibodies against pgsA were performed (A in FIG. 11). In the figure, lane 1 is untransformed live Lactobacillus casei, lane 2 is transformed pHCE1LB: pgsA-P5 / transfected Lactobacillus casei, lanes 3 and 4 are transformed pHCE1LB: pgsA-Anal3 / live Lactobacillus Bacillus casei, lane 5 shows untransformed dead bacterium (heat treated) Lactobacillus casei, lane 6 transformed pHCE1LB: pgsA-P5 / bacterial (heat treated) Lactobacillus casei, le 7 is a transformant pHCE1LB: pgsA-Anal3 / dead cells (heat treatment) is Lactobacillus casei.
    [89] As shown in FIG. 11B, it can be seen that pgsA, peptide antibiotic P5, and Anal3 are present in the same fusion protein form as before the heat treatment.
    [90] (2) The activity of plasmid in Lactobacillus sterilized by heat treatment was confirmed.
    [91] As in the above-described example, transformation with the surface expression vectors pHCE1LB: pgsA-P5 and pHCE1LB: pgsA-Anal3, respectively, which were found to express fusion proteins of antibiotic peptide P5 fused with pgsA and Anal3 fused with pgsA, respectively Lactobacillus was used. The transformed lactobacillus was heat-treated at 110 ° C. for 20 minutes, and then plasmids in the heat-treated lactobacillus were confirmed (B in FIG. 11): in FIG. 1, lane 1 was a live cell lactose transformed with pHCE1LB: pgsA-P5. Bacillus casei, lane 2 is heat-treated Lactobacillus casei transformed with pHCE1LB: pgsA-P5, lane 3 is live bacterium Lactobacillus casei transformed with pHCE1LB: pgsA-Anal3, lane 4 is transformed with pHCE1LB: pgsA-Anal3 Converted bacterium (heat treated) Lactobacillus casei.
    [92] As can be seen in the figure, it can be seen that all of the plasmids were denatured or denatured by heat treatment.
    [93] Although not shown as separate data, when transformed to E. coli plasmid extracted after the heat treatment in the same manner as above did not show any transformed E. coli. Therefore, the transformed microorganism according to the present invention was confirmed that the proper heat treatment can prevent the heterogeneity of genes or movement between organisms through the plasmid.
    [94] Indirect Example: Construction of a vector inserted with a combination of pgsB, pgsC and pgs A and foreign protein surface expression experiment using the same
    [95] One or two or more of the genes pgsB, pgsC, and pgsA encoding the polygammaglutamic acid synthase complex and the peptide antibiotics according to the present invention can be prepared, but the surface expression vector containing the gene encoding the foreign protein can be prepared. In addition, by transforming these vectors into microorganisms, it was confirmed that foreign proteins were expressed on the surface of the microorganisms.
    [96] Thereby, any one or two or more of the genes pgsB, pgsC and pgsA encoding the polygammaglutamic acid synthase complex, and the gene encoding the amphiphilic peptide antibiotic having antibacterial, antifungal and anticancer activity according to the present invention It is indirectly confirmed that antibiotic surface expression vectors can be produced and used for surface expression.
    [97] In an indirect embodiment, the plasmids pGNBCA and pGNCA are identical to the plasmids pGNpgsBCA and pGNpgsCA in the present invention, respectively.
    [98] (Indirect Example 1) Preparation of Conversion Vector pGNBCA-HB168 for Surface Expression and Surface Expression of Neutralizing Antibody-forming Antigen Group of S Antigen
    [99] Conversion vector capable of surface expression of neutralizing antibody-forming antigenic group of hepatitis B virus S antigen using Gram-negative microorganism as a host by using gene of extracellular membrane protein (pgsBCA) involved in polygammaglutamic acid synthesis derived from Bacillus sp. pGNBCA-HB168 was prepared.
    [100] In order to introduce the hepatitis B virus S antigen gene into the surface expression vector pGNBCA which is a Gram-negative microorganism host, about 1.4 kb of hepatitis B virus gene cloned in the general cloning vector pUC8 is used as a template, Polymerization using an oligonucleotide having the nucleotide sequence of 8 (5-ctg gga tcc caa ggt atg ttg ccc gtt tg-3) and SEQ ID NO: 9 (5-tga agc tta tta gga cga tgg gat ggg aat-3) as a primer Enzyme chain reaction was performed to amplify the S antigen gene. The amplified gene region was 168 bp in size.
    [101] The primers of SEQ ID NO: 8 and SEQ ID NO: 9 were constructed such that restriction sites BamH I and Hind III recognition sites present in the surface expression vector pGNBCA were present. Translation codon is applied to the C-terminal region of the gene of the extracellular membrane protein involved in polygammaglutamic acid synthesis of the surface expression vector pGNBCA prepared in advance by cutting the amplified hepatitis B virus S antigen gene with restriction enzymes BamH I and Hind III. Connected accordingly. The conversion vector pGNBCA-HB168 thus prepared is shown in FIG. 12.
    [102] The surface expression of the neutralizing antibody-forming antigenic group of hepatitis B virus S antigen in Escherichia coli was investigated using the surface expression vector pGNBCA-HB168.
    [103] E. coli was transformed into the expression vector prepared in Example 2, and then 50 ml of Elbi medium (5 g / L yeast extract, 10 g / L tryptone, 5 g / salt) to which 100 mg / L of antibiotic (ampicillin) was added. Surface expression was induced by growth in a 500 ml flask containing L, pH 7.0).
    [104] Bacterial expression of the neutralizing antibody-forming antigenic group of the S antigen fused with the C-terminus of the gene synthesizing polygammaglutamic acid was performed by SDS-polyacrylamide gel electrophoresis and western blotting using antibodies against the S antigen. It was confirmed by performing. Specifically, samples were prepared by denatured proteins obtained at the same cell concentration, analyzed by SDS-polyacrylamide gel electrophoresis, and the fractionated proteins were transferred to PVDF membranes. The PVDF membrane to which the proteins were transferred was blocked by blocking for 1 hour in blocking buffer (50 mM Tris hydrochloric acid, 5% skim milk, pH 8.0), and then the polyclonal primary antibody derived from sheep against S antigen was blocked. The solution was diluted 1000-fold and reacted for 12 hours. After completion of the reaction, the membrane was washed with a buffer solution, and the biotin-conjugated secondary antibody was diluted 1000-fold in the blocking buffer and reacted for 4 hours. After completion of the reaction, the membrane was washed with a buffer solution and washed again by reacting avidin-biotin reagent for 1 hour. The washed membrane was colored by adding H 2 O 2 and DAB solution as a substrate and a color developing reagent, and confirmed specific binding between the specific antibody to the S antigen and the fusion protein (FIG. 13A). In FIG. 13A, lane 1 is untransformed host cell JM109, and lane 2 is transformed pGNBCA-HB168 / JM109. As shown in the figure, the fusion protein band of about 48 KDa was confirmed by pGNBCA-HB168 plasmid.
    [105] In addition, SDS-polyacrylamide gel electrophoresis and separation of the soluble, innermembrane, outermembrane, etc. of E. coli induced expression by the outeremembrane fractionation method to directly confirm that the antigen-forming antigen group of the S antigen is expressed on the surface of E. coli. Western blotting using antibodies against the S antigen was performed. Specifically, E. coli, the surface expression of the fusion protein induced by the method described above, was harvested to the same cell concentration as E. coli untransformed, and the cells were washed several times with a buffer solution (10 mM HEPES buffer, pH 7.4), / Ml lysozyme, 1 mM PMSF and 1 mM EDTA and suspended in a buffer solution and reacted for 10 minutes at 4 ℃, then added DNase (0.5 mg / ㎖) and RNase (0.5 mg / ㎖) by sonication After cell destruction, Intact Escherichia coli and cellular debris were separated by 10,000 X g centrifugation at 4 ° C. for 20 minutes, and the isolated E. coli cellular debris were separated by 15,000 X g centrifugation at 4 ° C. for 2 hours. Fractions containing proteins of periplasm and cytoplasm were obtained. The obtained pellet was suspended in a buffer solution containing 1% Sarcosyl (N-lauryl sarcosinate, sodium salt) (PBS, pH 7.4), and then the supernatant was separated by 15,000 X g centrifugation at 4 ° C for 2 hours. As an inner membrane, pellets were obtained from E. coli outermembrane protein, and each fraction was subjected to SDS-polyacrylamide gel electrophoresis and Western blotting using an antibody against S antigen. It was confirmed that the neutralizing antibody-forming antigen group was located on the outer membrane ( FIG. 13A ; E. coli membrane fraction Western blot result). In FIG. 13A, lane 1 is untransformed JM109, lane 2 is a whole cell of transformed pGNBCA-HB168 / JM109, lane 3 is a soluble fraction of transformed pGNBCA-HB168 / JM109, and lane 4 is transformed pGNBCA- Innermembrane fraction of HB168 / JM109, lane 5 shows the outermembrane fraction of transformed pGNBCA-HB168 / JM109.
    [106] Fluorescence-activating cell sorting (FACS) flow cytometry confirmed that the expression of the neutralizing antibody-forming antigenic group of S antigen was expressed on the surface of E. coli by the C-terminus of polygammaglutamic acid synthesis protein. . For immunofluorescence staining, E. coli, which induced expression, was harvested to the same cell concentration, and the cells were washed several times with buffer solution (PBS buffer, pH 7.4), and then buffered solution containing 1% bovine albumin 1 The cells were suspended in mL and diluted 1000-fold from the polyclonal primary antibody derived from sheep against S antigen, and reacted at 4 ° C for 12 hours. After completion of the reaction, the cells were washed several times with a buffer solution, suspended in 1 ml of a buffer solution containing 1% bovine serum albumin, and the biotin-bound secondary antibody was diluted 1000-fold and reacted at 4 ° C. for 3 hours. The cells were then washed several times with buffer, suspended in 0.1 ml of buffer containing 1% bovine serum albumin, and then streptoavidin-R-phycoerythrin staining reagent specific for biotin. Was bound by diluting 1000 fold.
    [107] E. coli was washed several times after the reaction and measured by fluorescence-activated cell screening method, it was confirmed that the neutralizing antibody-forming antigen-based protein of the S antigen compared to the non-transformed E. coli is expressed on the surface (Fig. 13B). In FIG. 13B, white indicates untransformed JM109 and black indicates derived from transformed pGNBCA-HB168 / JM109. As shown in the figure, the S antigen neutralizing antibody former was not expressed in E. coli that was not transformed, but the surface expression of the S antigen neutralizing antibody former of E. coli transformed by the surface expression vector was clearly confirmed.
    [108] (Indirect Example 2) Preparation of Switching Vector pGNCA-HB168 for Surface Expression and Formation of Neutralizing Antibody-forming Antigen Group of Hepatitis B Virus S Antigen
    [109] A vector for surface expression using a gram-negative microorganism using pgsC and pgsA among genes of the extracellular membrane protein (pgsBCA) involved in polygammaglutamic acid synthesis derived from a Bacillus strain was prepared.
    [110] To obtain genes encoding the N-terminus and C-terminus of pgsC and pgsA among the extracellular membrane proteins involved in polygammaglutamic acid synthesis, the total chromosome was used as a template and sequence 10 (5-gca cat atg) was used at the N-terminus. ttc gga tca gat tta tac atc-3), polymerase chain reaction using an oligonucleotide having the nucleotide sequence of SEQ ID NO: 11 (5-ctc gga tcc ttt aga ttt tag ttt gtc act-3) as a primer Was performed.
    [111] In SEQ ID NO: 10, the primer for the N-terminus, the restriction enzyme Nde I recognition site present in the expression vector pHCE 19T (II) was present. The amplified gene region was about 1.6 kb in size from the N-terminal region of pgsC, the extracellular membrane protein gene involved in polygammaglutamic acid synthesis, to the C-terminal region of pgsA.
    [112] Genes amplified by the polymerase chain reaction were digested with restriction enzymes Nde I and BamH I and inserted into pHCE 19T (II), a constant high-expression vector already digested with Nde I and BamH I, to participate in polygamma glutamic acid synthesis. A new approximately 5.3 kb vector was prepared with no translation end codon added to the gene of the extracellular membrane protein and a new restriction enzyme recognition site, and named pGNCA.
    [113] PgsC and pgsA of genes of the extracellular membrane protein (pgsBCA) involved in polygamma glutamic acid synthesis using pgsC and pgsA to convert gram-negative microorganisms into the host vector pGNCA- HB168 was produced in the same manner as in the previous Example 2. The conversion vector pGNCA-HB168 thus prepared is shown in FIG. 14.
    [114] Expression of the neutralizing antibody-forming antigenic group of hepatitis B virus S antigen in E. coli was investigated using the surface expression vector pGNCA-HB168.
    [115] To this end, the expression vector was transformed into Escherichia coli and the expression was induced in the same manner as in Example 3, followed by SDS-polyacrylamide gel electrophoresis that the neutralizing antibody antigen group of the S antigen fused with the extracellular membrane protein pgsCA was expressed in E. coli. Confirmation was performed by Western blotting with antibodies to the S and antigens.
    [116] In FIG. 15A, lane 1 is untransformed host cell JM109, and lane 2 is transformed pGNCA-HB168 / JM109. As shown in the figure, the fusion protein band of about 48 KDa was identified by pGNCA-HB168 plasmid.
    [117] As described and demonstrated in detail above, the present invention provides all of the genes of the extracellular membrane protein (pgsBCA) involved in the synthesis of poly-gamma glutamic acid derived from Bacillus sp. As a surface-expressing matrix to efficiently express foreign proteins on the surface of microorganisms. Alternatively, the present invention provides a surface expression vector capable of expressing the peptide antibiotics P5 and Anal3 on the cell surface with an affinity structure that exhibits antimicrobial, antifungal and anticancer activity.
    [118] According to the present invention, a method for efficiently expressing the peptide antibiotic on the surface of the transformant in various transformants transformed with the surface expression vector and the effective use of the transformant for surface expression of the peptide antibiotic are possible. Done.
    [119] Peptide antibiotic surface expression of the present invention provides a large amount of peptide antibiotics inexpensively by using lactic acid bacteria, which have been proven safe and effective in the pharmaceutical and food fields, without the purification process of peptide antibiotics P5 and Anal3. The possibility of industrial application is very high. In addition, other peptide antibiotics other than peptide antibiotics P5 and Anal3 can be applied through the present technology.
    权利要求:
    Claims (16)
    [1" claim-type="Currently amended] Antibiotic surface expression vector comprising any one or more of genes pgsB, pgsC and pgsA encoding polygammaglutamic acid synthase complex and gene encoding an affinity peptide antibiotic having antibacterial, antifungal and anticancer activity .
    [2" claim-type="Currently amended] The method of claim 1,
    An antibiotic surface expression vector comprising only pgsA among genes encoding the polygammaglutamic acid synthase complex.
    [3" claim-type="Currently amended] The method according to claim 1 or 2,
    The antimicrobial peptide antibiotic having antimicrobial, antifungal and anticancer activity is an antibiotic surface expression vector, characterized in that it is identical to peptide P5 encoded by the nucleotide sequence of SEQ ID NO: 4.
    [4" claim-type="Currently amended] The method of claim 3, wherein
    The vector is a surface expression vector for antibiotics, characterized in that pHCE1LB: pgsA-P5 which can be expressed on the surface of Gram-negative and positive bacteria.
    [5" claim-type="Currently amended] Microorganism transformed with the vector pHCE1LB: pgsA-P5 according to claim 4.
    [6" claim-type="Currently amended] Escherichia coli transformed with the vector pHCE1LB: pgsA-P5 according to claim 4 (KCTC 10350BP).
    [7" claim-type="Currently amended] Lactobacillus casei transformed with the vector pHCE1LB: pgsA-P5 according to claim 4.
    [8" claim-type="Currently amended] A pharmaceutical composition for antimicrobial, antifungal or anticancer activity comprising the Lactobacillus casei according to claim 7, wherein the peptide antibiotic P5 is surface-expressed by culture, or a Lactobacillus suspension containing peptide antibiotic P5 as an active ingredient.
    [9" claim-type="Currently amended] The method of claim 8,
    Antimicrobial, antifungal, or anticancer pharmaceutical composition, characterized in that the heat treatment of the active ingredient.
    [10" claim-type="Currently amended] The method according to claim 1 or 2,
    The antimicrobial peptide antibiotic having antimicrobial, antifungal and anticancer activity is an antibiotic surface expression vector, characterized in that it is identical to peptide Anal3 encoded by the nucleotide sequence of SEQ ID NO: 6.
    [11" claim-type="Currently amended] The method of claim 10,
    The vector is a surface expression vector for antibiotics, characterized in that pHCE1LB: pgsA-Anal3 that can be expressed on the surface of Gram-negative and positive bacteria.
    [12" claim-type="Currently amended] A microorganism transformed with the vector pHCE1LB: pgsA-Anal3 according to claim 11.
    [13" claim-type="Currently amended] Escherichia coli transformed with the vector pHCE1LB: pgsA-Anal3 according to claim 11 (KCTC 10348BP).
    [14" claim-type="Currently amended] Lactobacillus casei transformed with the vector pHCE1LB: pgsA-Anal3 according to claim 11.
    [15" claim-type="Currently amended] A pharmaceutical composition for antimicrobial, antifungal or anticancer activity comprising the Lactobacillus casei according to claim 14 or the Lactobacillus suspension containing peptide antibiotic P5 as an active ingredient.
    [16" claim-type="Currently amended] The method of claim 15,
    Antimicrobial, antifungal, or anticancer pharmaceutical composition, characterized in that the heat treatment of the active ingredient.
    类似技术:
    公开号 | 公开日 | 专利标题
    Klauser et al.1990|Extracellular transport of cholera toxin B subunit using Neisseria IgA protease beta‐domain: conformation‐dependent outer membrane translocation.
    Dombek et al.1999|High‐frequency intracellular invasion of epithelial cells by serotype M1 group A streptococci: M1 protein‐mediated invasion and cytoskeletal rearrangements
    Wells et al.1996|Lactic acid bacteria as vaccine delivery vehicles
    US5508192A|1996-04-16|Bacterial host strains for producing proteolytically sensitive polypeptides
    ES2335136T3|2010-03-22|Fagical expression through cotraductional translocation of fusion polypeptides.
    Wood et al.1996|SopE, a secreted protein of Salmonelladublin, is translocated into the target eukaryotic cell via a sip‐dependent mechanism and promotes bacterial entry
    EP1066375B1|2004-12-29|$i| HARBORING AGGREGATION AND MUCIN BINDING GENES AS VACCINE DELIVERY VEHICLES
    JP3753945B2|2006-03-08|Plasmid shuttle vector between Escherichia coli and Brevibacillus bacteria
    DE60215461T2|2007-05-03|Serpin of bifidobakteria
    De Grado et al.1999|Identification of the intimin‐binding domain of Tir of enteropathogenic Escherichia coli
    EP1808489B1|2016-03-23|Expression system for Bacillus anthracis PA
    CN1325644C|2007-07-11|Muteins of human neutrophil gelatinase-associated lipocalin and related proteins
    US7858357B2|2010-12-28|Immunization with bacterial ghost-based vaccines
    TW514667B|2002-12-21|Methods for production of recombinant plasmids
    CA2116914C|2005-02-08|Novel plasmid for production of crm protein and diphtheria toxin
    Hynönen et al.2002|Identification by flagellum display of an epithelial cell-and fibronectin-binding function in the SlpA surface protein of Lactobacillus brevis
    Ebel et al.1998|Initial binding of Shiga toxin‐producing Escherichia coli to host cells and subsequent induction of actin rearrangements depend on filamentous EspA‐containing surface appendages
    EP1328628B1|2008-12-31|Method for exposing peptides and polypeptides on the cell surface of bacteria
    Yamamoto et al.2003|Localization of the vegetative cell wall hydrolases LytC, LytE, and LytF on the Bacillus subtilis cell surface and stability of these enzymes to cell wall-bound or extracellular proteases
    US5652116A|1997-07-29|Method for genetically manipulating peptide synthetases
    AU729449B2|2001-02-01|Materials and methods relating to the attachment and display of substances on cell surfaces
    Oxaran et al.2012|Pilus biogenesis in Lactococcus lactis: molecular characterization and role in aggregation and biofilm formation
    Callegari et al.1998|The S-layer gene of Lactobacillus helveticus CNRZ 892: cloning, sequence and heterologous expression
    AU720857B2|2000-06-15|Use of toxin peptides and/or affinity handles for delivery compounds into cells
    de Marsac et al.1987|Expression of the larvicidal gene of Bacillus sphaericus 1593M in the cyanobacterium Anacystis nidulans R2
    同族专利:
    公开号 | 公开日
    KR100525759B1|2005-11-03|
    引用文献:
    公开号 | 申请日 | 公开日 | 申请人 | 专利标题
    法律状态:
    2002-10-17|Application filed by 한국생명공학연구원, 주식회사 바이오리더스, 학교법인조선대학교
    2002-10-17|Priority to KR10-2002-0063379A
    2004-04-29|Publication of KR20040034780A
    2005-11-03|Application granted
    2005-11-03|Publication of KR100525759B1
    优先权:
    申请号 | 申请日 | 专利标题
    KR10-2002-0063379A|KR100525759B1|2002-10-17|2002-10-17|Surface - expression method for peptides P5 and Anal3 using pgs A|
    [返回顶部]