Recombinant combo protein fused in HIV type-1, -2 and 0, manufacturing method thereof, vector and tr

专利摘要:
The present invention provides a recombinant Combo protein fused to a human immunodeficiency virus (hereinafter referred to as 'HIV')-1, -2, O type protein, a method for producing the same, and a vector comprising the same. The present invention relates to a transformed Escherichia coli, a diagnostic kit, and a composition for a vaccine. An expression vector is prepared to express high antigenic proteins of HIV-1, -2, and -O as one protein (Combo protein), The transformed Escherichia coli was amplified by the T7 / lac promoter, and expressed by attaching a histidine residue to the N-terminus of the target protein, ie, a combo protein, to form a target protein with a cation histidine. Probond) is a method for producing a large amount of HIV combo antigen can be purified by an easy method of adsorption on the column.
公开号:KR20030060274A
申请号:KR1020020000896
申请日:2002-01-08
公开日:2003-07-16
发明作者:손미진；김은경；유승범；김형철；이상익；오재훈
申请人:주식회사 엘지생명과학；
IPC主号:

专利说明:

Recombinant combo protein fused in HIV type-recombinant combo protein fused with human immunodeficiency virus-1, -2, 0 type protein, production method thereof, vector and transformed E. coli, diagnostic kit and vaccine comprising same 1, -2 and 0, manufacturing method according, vector and transformant specifically, diagnostic kit according and composition for vaccine
[7] The present invention provides a recombinant Combo protein fused to a human immunodeficiency virus (hereinafter referred to as 'HIV')-1, -2, O type protein, a method for producing the same, and a vector comprising the same. It relates to a composition for transformed E. coli, diagnostic kit and vaccine.
[8] Human Immunodeficiency Virus (HIV) is largely classified into two types, HIV-1 and HIV-2, and HIV-1 is again divided into two groups, group M and group O. Classified as Group M is the most common worldwide, and group O is mainly distributed in western Africa. However, patients infected with this group O are increasingly being identified in Europe and the United States. In addition, HIV-2 type is mainly found in West Africa, but is gradually spreading around the world (Charneau P. et al., Virology, 205, 247-53, 1994; Gould K. et al., Lancet , 348, 680-1, 1996; Quinn TC, Proc Natl Acad Sci USA, 91, 2407-14, 1994).
[9] Although these three types have a common epitope, they are often unable to detect HIV-2 type as a diagnostic reagent for HIV-1 type due to low cross-reactivity (Clavel F. et al. , Nature , 324, 691-5, 1986; Schable C. et al., Lancet, 344, 1333-4, 1994), with the recent import of a large number of foreign blood in Korea, especially for the accurate diagnosis of blood screening for the HIV-1 group. There was a need for a diagnostic reagent capable of diagnosing both M, HIV-1 group O, and HIV-2 type.
[10] One of the targets for diagnosing HIV infection is the glycoprotein gp41, which encodes the envelope gene ( env ) of the viral gene (Gnann JW Jr. et al ., J. Virol ., 61, 2639-41, 1987). Sex is very high and antibodies to this protein persist strongly throughout the entire process of Acquired Immuno-deficiency Syndrome (hereinafter referred to as AIDS) (Weiss RA, Science 260, 1273-9, 1993). However, due to the nature of the very hydrophobic ( env gp41) protein, there was a technical difficulty in expressing only a large amount of the env gene in E. coli.
[11] In order to solve this problem, existing methods, such as env gene, beta-galactosidase (β-gal), maltose binding protein, GST gene, etc. fused to increase the solubility and improve the expression rate Expression was attempted (Ellinger S. et al., Virology , 180, 811-3, 1991; Morimoto M. et al . , AIDS Res. Hum.Retroviruses, 9, 971-8, 1993). However, even in such a method, since the heterologous fusion protein is added, there has been a problem that it must be removed after purification.
[12] In order to solve the above problems, an object of the present invention is to provide an expression vector, cell line, and protein for expressing a fusion protein having a known specificity for HIV type and excellent antigenicity.
[13] In addition, an object of the present invention is to provide a diagnostic kit or vaccine production composition for diagnosing HIV infection comprising a fusion protein having specificity for each HIV type and excellent antigenicity.
[1] 1 shows primers synthesized for the preparation of plasmid pLCSK-1 expressing the Combo protein (SEQ ID NO: 1 to 6).
[2] Figure 2 depicts the amino acid sequence of the combo protein (SEQ ID NOs: 7-11).
[3] Figure 3 is a flow chart showing the manufacturing process of the plasmid pLCSK-1 expressing the HIV combo protein in E. coli.
[4] Figure 4 is a photograph showing the purified pattern of the combo protein using a Probond adsorbent resin as a result of SDS-polyacrylamide gel electrophoresis.
[5] 5 is a photograph showing Western blot of HIV-1, -2, O type positive patient serum to assay the antigenicity of the combo protein.
[6] FIG. 6 is a graph showing the results of specificity sensitivity determination of enzyme immunity measurement using purified combo antigens from serum of positive and negative patients of HIV-1, -2, O type and serum of normal persons.
[14] In order to achieve the above object, the present invention, human immunodeficiency virus (hereinafter referred to as "HIV")-1, -2, O of the antigen of the virus of type O, the high HLZaaijer et al. (J. Med. Viro., 51: 80-82, 1997) and Robin A. Weiss et al. (Science, vol. 260, 1273-1278, 1993). 1 A portion of the antigenic protein gp41, a portion of the HIV-2 antigen protein gp36, a mutant type of the HIV-1 O-type antigen protein gp41 and the HIV-1 antigen protein gag p24 protein It provides an expression vector pLCSK-1 for E. coli and an expression cell line BL21 (DE3) / pLCSK-1 including the same, which can induce the expression of each virus type antigen as a chimeric protein.
[15] The present invention also allows the expression vector to repeat the codon encoding histidine six times at the end of the gene sequence encoding the four HIV-derived fusion proteins, with the characteristics of antigen solubility and expression rate improvement when inducing protein expression The histidine residue is located at the C-terminus, and is a protein purification method using a commercially available 6 × His tag, Probond (Invitrogen Inc., US), catalog number R801-01. By using a single purification method) provides a method for producing a large amount of recombinant HIV Combo protein having several antigens at the same time.
[16] More specifically, the present invention includes a single or all of HIV type-1 p24 or gp41, HIV type-2 gp36, and HIV type-1 group O gp41. Provided are fusion proteins that are strand polypeptides.
[17] In addition, the present invention the HIV type-1 p24 protein comprises all or part of SEQ ID NO: 7; Said HIV type-1 gp41 protein comprises all or part of SEQ ID NO: 8; Said HIV type-2 gp36 protein comprises all or part of SEQ ID NO: 9; And the HIV type-1 group O gp41 protein provides the fusion protein, which comprises all or part of SEQ ID NO: 10.
[18] The present invention also provides the fusion protein, characterized in that six times histidine residues are located at the C-terminus of the fusion protein.
[19] The present invention also provides an expression vector pLCSK-1 encoding the fusion protein.
[20] In another aspect, the present invention provides a transformed Escherichia coli (Accession Number: KCTC 10100BP) characterized in that it comprises the expression vector.
[21] In another aspect, the present invention is a method for producing a fusion protein comprising a culture step, cell collection step, cell disruption step, separation step and purification step, characterized in that to produce a fusion protein using the transformed E. coli of claim 5 It provides a method for producing a fusion protein.
[22] In another aspect, the present invention provides a method for producing the fusion protein, characterized in that the purification step is made by adsorbing and separating six histitin residues at the C terminus of the fusion protein to the probond column.
[23] In another aspect, the present invention provides a diagnostic kit for HIV, characterized in that using the protein produced by the above method as an antigen.
[24] In another aspect, the present invention provides a vaccine composition comprising a protein produced by the above method.
[25] In the present invention, a peptide of HIV-1 type gp41 and HIV-2 type gp36 and gp41 of HIV-1 group O are linked to form a chimeric protein and its N-terminus. The solubility and expression of HIV gag peptide p24 protein, which is highly soluble, are linked to induce expression, thereby improving the solubility and expression rate of these antigens (see Table 1).
[26] In addition, six histidines that do not affect antigenicity and protein expression are attached to the C-terminus to provide HIV-1 p24 / HIV-1 gp41 / HIV-2 gp36 peptide / HIV-1 group O gp41. By making a recombinant protein fused with E. coli and expressing it in E. coli, a simple one-step method in which a target protein having a cation histidine is adsorbed and purified on a Probond column with nickel anion is attached. Since the HIV combo antigen is manufactured to be purified, the purification method can be improved.
[27] As a result, the expression level was also very high, and Western blotting was performed with the serum of AIDS patients infected with HIV-1 type, HIV-2 type and O-type, respectively. It was confirmed that the Merrick HIV combo antigen had antigenicity in all blood.
[28] In addition, since it does not contain heterologous proteins, it may have higher specificity than diagnostic reagents using antigens containing conventional heterologous fusion proteins.
[29] Therefore, the present invention can secure a large amount of antigens having higher specificity and sensitivity in the future by a simple purification method, so that the present invention can be usefully used for diagnostic reagents and vaccines for diagnosing HIV type-1, -2, and -O.
[30] In addition, the immunospecific antigen thus prepared can be mixed with a pharmaceutically acceptable carrier as an active ingredient to prepare a prophylactic or therapeutic vaccine injection composition.
[31] Hereinafter, the present invention will be described in detail by way of examples. However, the following examples are merely illustrative of the present invention and do not limit the scope of the present invention.
[32] <Preparation stage> Securing genes for HIV antigen
[33] (1) Securing the gene for HIV antigen (HIV-1 p24)
[34] Plasma was taken from the blood of an HIV-1 patient and from this the total RNA was separated therefrom. The reagent used at this time is a TRI reagent (Sigma, USA, catalog number T9424). The solution is mixed with the patient's plasma and centrifuged to obtain only an RNA layer. Prepanol (isopropanol) was precipitated, washed with ethanol (ethanol), and then dissolved in tertiary distilled water again.
[35] From the isolated RNA, cDNA was synthesized using an N6 primer (Genotech Co., Ltd.). In this process, cDNA was synthesized using a reverse transcriptase called SuperScript II (manufactured by Life Technologies, United States, catalog number 18064-022).
[36] PCR was performed using the obtained cDNA as a template using Primer 1 and Primer 2 of FIG. 1. PCR was performed 35 cycles in the order of 94 ℃, 50 ℃, 72 ℃, the enzyme was Vent DNA polymerase (New England Biolabs, United States, Catalog No. 254S) was used as a DNA machine (DNA engine, model No. PTC-200) manufactured by MJ Research, Inc., USA. Electrophoresis was performed on an agarose gel to confirm that DNA fragments of about 700bp were obtained. The obtained DNA fragment was purified with phenol / chloroform and treated with Nde I and BamH I.
[37] (2) Securing genes for HIV antigens (HIV-1 gp41 ', HIV-2 gp36')
[38] CDNA was used as a template for obtaining the gene of HIV-1 p24, and PCR was performed using primers 3 and 4 of FIG. At this time, PCR conditions, enzymes and machines used were the same as those for amplifying the gene of HIV-1 p24. Primer 4 was constructed to include a base sequence encoding HIV-2 gp36 '. It was electrophoresed on an agarose gel to confirm that DNA fragments of about 420 bp were obtained. The obtained DNA fragment was purified with phenol / chloroform and treated with BamH I and EcoR I.
[39] (3) Securing the gene for HIV antigen (HIV-O gp41 ')
[40] Plasma was taken from patient blood of HIV-O and total RNA was isolated therefrom. At this time, TRI reagent, isopropanol, ethanol and the like were used as in the case of obtaining the gene of HIV-1 p24. From the isolated RNA, cDNA was synthesized using an N6 primer (Genotech, Daejeon, Korea).
[41] Using primers 5 and 6 of Fig. 1, PCR was performed using the obtained cDNA as a template. Electrophoresis was performed on an agarose gel to confirm that DNA fragments of about 320bp were obtained. The obtained DNA fragment was purified with phenol / chloroform and treated with EcoR I and Xho I.
[42] Example 1 Preparation of recombinant expression vector pLCSK-1 and transformation of strain
[43] pET23a (Novagen, USA, catalog number 69745-3) was treated with Nde I and Xho I and subjected to ligation reaction with the three kinds of DNA fragments obtained in the above process. It was. The enzyme used at this time was a T4 DNA ligase (Roche, USA, catalog No. 716 359), which was transformed after an overnight reaction at 16 ° C. Was performed. An expression vector having a size of about 5.1 kb was confirmed from the obtained transformant, which was called pLCSK-1 (FIG. 3).
[44] The expression vector thus obtained was transformed into Escherichia coli BL21 (DE3) (Studier FW and Moffatt BA, J. Mol. Bio ., 189 (1), 113-30, 1986).
[45] The transformants were incubated overnight in LB medium supplemented with ampicillin (50 mg / ml), and the recombinant expression strain BL21 (DE3) / pLCSK-1 selected therefrom was deposited as a strain in the gene bank of the Biotechnology Research Institute (Accession No. KCTC10100BP).
[46] Example 2 Expression and Purification of HIV Combo Proteins in Recombinant Strains
[47] The deposited recombinant expression strain BL21 (DE3) .pLCSK-1 (Accession No. KCTC10100BP) was inoculated in 3 ml of LB medium to which ampicillin (50 mg / ml) was added, and shaken at 37 ° C. to obtain an absorbance of 0.8 nm at 600 nm. (Spectrophotometer was used Novaspec II visible (Amersham-Pharmacia, US Catalog No. 80-2088-64) at 600nm) When the absorbance reached 0.8, IPTG (Isopropylthiogalactoside) was added so as to have a final concentration of 1 mM, and further cultured for 4 hours.
[48] Centrifugation of 1ml of the culture solution to recover only the cells, and then redispersed by adding PBS (Phosphate-buffered saline), pH 7.4, 100μl, 50μl of SDS-sample buffer (additional) at 100 ℃ Heated for 5 minutes. This was centrifuged at 14,000 rpm for 10 minutes to recover only the supernatant. Thereafter, 12% SDS-PAGE (Laemmli UK, Nature 227, 680-5, 1970) was performed to confirm that the expression of the protein at the molecular weight of 50 kDa was strongly expressed (FIG. 4).
[49] For protein purification, recombinant expression strain BL21 (DE3) .pLCSK-1 (Accession No. KCTC 10100 BP) was inoculated in 30 ml of LB medium supplemented with ampicillin (50 mg / ml), and shaken at 37 ° C. for 18 hours. Saturated cultures were inoculated in 1 L of the same LB medium and shaken at 37 ° C until the absorbance at 600 nm was 0.8. When the absorbance at 600 nm reached 0.8, IPTG (Isopropylthiogalactoside) was added so as to have a final concentration of 1 mM, and further cultured for 4 hours. Centrifuge 1 L of this shake culture solution (6,000 rpm, 4 ° C., 20 minutes, Beckman Coulter, Inc., USA), Beckman J2-21M Centrifuge, 2 X 500 ml, Catalog No. 344301), the culture medium was discarded, and only the cells were taken and redispersed in 30 ml of dispersion (PBS, pH 7.4). The dispersed cells were crushed using an ultrasonic crusher (Branson, USA, product name Sonyfire 450).
[50] Since the expressed HIV combo protein contains a hydrophobic site, it exists in the zone of centrifuged sediment, so it is centrifuged (14,000 rpm, 4 ° C., 30 minutes, Hitachi Ltd., Japan, product name himac). CR 22F, 50 ml, Cat. No. S101521B), the supernatant was discarded, and the remaining precipitate was dissolved in 6M guanidine-HCl to collect a solution containing HIV combo protein.
[51] Subsequently, for purification of the HIV combo protein, a Probond column (Invitrogen Inc., USA) using six histidine residues that were expressed at the C-terminus of the HIV combo protein upon protein expression. And catalog number R801-01). Unadsorbed impurities are removed by washing with PBS (pH 7.4) containing 6M guanidine-HCl and 20 mM imidazol, and the adsorbed surface antigens are 6M guanidine-HCl and 300 mM imidazole. It was separated by desorption with PBS (pH 7.4).
[52] In this process, the surface antigen was found to be more than 95% pure separation from the molecular weight of 50Kd SDS PAGE results (Fig. 4). This separation solution was dialyzed with 8M urea and used as a component for various uses including diagnostic kits.
[53] Example 3 Antigen Assay of HIV Combo Protein Purified in Escherichia Coli
[54] In Example 2, 12% SDS-PAGE was performed to confirm the expression of the HIV combo protein having a molecular weight of 50 Kd, and Western blotting was performed with AIDS patient serum to test the antigenicity of the protein.
[55] After the cell lysate induced by the expression of the HIV combo protein or the purified HIV combo protein solution was electrophoresed in 12% SDS-PAGE, the remaining gel was immersed in 25 mM Tris-HCl, 190 mM glycine (pH 8.3) for 30 minutes, After soaking in 20% methanol solution, the protein was transferred to nitrocellulose paper using the same buffer solution. Protein-transferred nitrocellulose paper was blocked with 1% Bovine Serum Albumin (BSA) / PBS, pH 7.4 buffer for 1 hour at room temperature, and diluted to 1/100 of each AIDS patient serum (HIV). -1 type, HIV-2 type, and HIV-1 group O type) were reacted at room temperature for 1 hour. Wash the finished paper with 0.05% Tween / PBS solution 5 times at 5 minute intervals, and then use HRP-conjugated anti-human goat secondary antibody conjugated with 1/2000 diluted goat. Vector Labs, Inc.) was reacted at room temperature for 1 hour. The nitrocellulose paper was then washed with 0.05% Tween / PBS solution and then developed with 4-chloro-naphthol and hydrogen peroxide (Towbin et al., Proc. Natl. Acad. Sci. USA, 76, 4350-4, 1979).
[56] As a result, HIV combo protein was detected by sera for three different HIV-subtypes, and all of them detected only a specific band of 50kDa, and thus the antigen specificity of HIV combo protein could be tested (Fig. 5).
[57] Example 4 Preparation of 96-plate for HIV Antibody Assay
[58] 0.1 M carbonate in a 96-well plate (Nunc, U.S.), a combo antigen solution purified in Example 2 for the enzyme immunoassay (Enzyme Immuno Assay, EIA) at a concentration of 1 µg / ml Diluted in buffer solution (pH 9.5) and dispensed 100ul. The plate was sealed and left at 4 ° C. for 18 hours to allow antigen to adhere to the wells. Thereafter, the antigen solution was removed, and 300 μl of PBS containing 0.1% BSA was dispensed per well, and left at 4 ° C. for 18 hours. The solution in the well (well) was removed and left at room temperature for 1 hour to dry the moisture, and then placed in a sealed container with a dehumidifying agent and stored in a 4 ℃ low temperature refrigerator and used if necessary.
[59] Example 5 Indirect EIA Assay for HIV Antibodies Using Combo Antigen
[60] The recombinant combo antigen obtained by the method of Example 2 was coated with a protein in a 96 well-plate in the same manner as in Example 4 to prepare a plate for use in assays, and the Negative plasma samples (samples) and antibody positive samples were added to each well 20µl, and 100µl of sample dilution solution (PBS buffer) was added and mixed well. The wells were washed five times with 300 μl of PBS containing Tween 20, and the reaction was performed at 37 ° C. for 30 minutes after adding a secondary antibody conjugated with peroxidase, followed by Tween 20. The wells were washed five times with 300 μl of PBS containing 100 μl of substrate solution (tetramethyl benzidine 100 μg / ml, hydrogen peroxide 0.006%, citric acid phosphate buffer pH 4.5). After 30 minutes of color development in the dark, 100 µl of the reaction stopper (2N sulfuric acid solution) was added to each well, and 96 nm reader (Molecular Devices, USA) was used as the reference wavelength of 650 nm. Absorbance was measured at 450 nm. Cut-off was determined by adding 0.2 to the average of negative samples, and 50 HIV PCR positive samples and 50 negative samples were tested by the above method. As a result, 50 out of 50 positive samples were determined to be positive, and 50 out of 50 positive samples were determined to be negative, showing 100% sensitivity and 100% high specificity (FIG. 6).
[61] Example 6 Sensitivity Comparison Experiment with Other Companies
[62] The titer of the combo antigen, ie sensitivity specificity, is shown in Table 1 below in comparison to the existing recombinant HIV antigens. The amount of antigen used in these experiments was approximately 1 μg / well, mostly carried out by enzyme immunoassay.
[63] antigen% Specificityresponsiveness(%)yieldRemarks P24 gag : 100-30910099.5-Ellinger et al., Virology, 180, 811 (1991) P121 Env : 439-64099.899.04mg / gGhrayeb et al., HIV detection by genetic Engineering Method, p41 Roche HIV gag / env 99.899.52.5-3mg / gShoeman et al., HIV detection by genetic Engineering Method, p99 Combo10010015-20mg / lThe present invention
[64] As shown in Table 1, the combo antigen of the present invention was found to be superior in specificity and sensitivity than in the case of using p24 or env protein alone and using a mixture of gag / env .
[65] As described above, the present invention is a gene that can be used as a diagnostic reagent or HIV vaccine composition for HIV infection due to high expression and easy purification, high sensitivity and specificity for antibodies compared to conventional antigen diagnostic targets It is a useful invention to provide a recombinantly expressed HIV combo protein, a vector comprising the same and transformed E. coli, and a method for producing the combo protein using the same.

权利要求:
Claims (9)
[1" claim-type="Currently amended] Human immunodeficiency virus (hereinafter referred to as 'HIV') type-1 p24 or gp41, HIV type-2 gp36, HIV type-1 group O gp41 A fusion protein that is a single-stranded polypeptide comprising a.
[2" claim-type="Currently amended] The method of claim 1,
Said HIV type-1 p24 protein comprises all or part of SEQ ID NO: 7;
Said HIV type-1 gp41 protein comprises all or part of SEQ ID NO: 8;
Said HIV type-2 gp36 protein comprises all or part of SEQ ID NO: 9; And
Said HIV type-1 group O gp41 protein comprises all or part of SEQ ID NO: 10.
[3" claim-type="Currently amended] The method of claim 2,
The fusion protein, characterized in that six times histidine residue is located at the C-terminus of the fusion protein.
[4" claim-type="Currently amended] The expression vector pLCSK-1 encoding the fusion protein of claim 3.
[5" claim-type="Currently amended] The method of claim 4, wherein
E. coli (transformation number: KCTC 10100BP) characterized in that it comprises the expression vector.
[6" claim-type="Currently amended] In the production method of the fusion protein comprising a culturing step, cell collection step, cell disruption step, separation step and purification step,
Method for producing a fusion protein, characterized in that to produce a fusion protein using the transformed E. coli of claim 5.
[7" claim-type="Currently amended] The method of claim 6,
Wherein the purification step is a method for producing the fusion protein, characterized in that by the method of separating by adsorbing six histitin residues at the C-terminal of the fusion protein to the probond column.
[8" claim-type="Currently amended] A diagnostic kit for HIV, which uses a protein produced by the method of any one of claims 6 and 7 as an antigen.
[9" claim-type="Currently amended] A vaccine composition comprising a protein produced by the method of any one of claims 6 and 7.

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同族专利:

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公开号 | 公开日

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KR100466382B1|2005-01-14|

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:
2002-01-08|Application filed by 주식회사 엘지생명과학

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2002-01-08|Priority to KR10-2002-0000896A

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2003-07-16|Publication of KR20030060274A

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2005-01-14|Application granted

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2005-01-14|Publication of KR100466382B1

优先权:

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KR10-2002-0000896A|KR100466382B1|2002-01-08|2002-01-08|Recombinant combo protein fused in HIV type-1 and -2, manufacturing method thereof, vector, transformant and diagnostic kit thereof|