KR100236765B1 - Immunodominant epitope of envelope 1 and envelope 2 protein in hepatitis c virus - Google Patents

Abstract

The present invention relates to antigenic determinants of envelope 1 and envelope 2 proteins of hepatitis C virus (hereinafter referred to as “HCV”).

More specifically provided by identifying the major immunodominant epitopes that specifically react with the serum of hepatitis C patients using envelope 1 and envelope 2 protein fragments expressed from the envelope 1 and envelope 2 genes of HCV. It relates to an antigenic protein, which has a short length and specifically reacts with the serum of a hepatitis C patient, and relates to an antigenic protein. The gene of the antigenic determinant of the envelope 1 or envelope 2 protein of HCV is combined with the ubiquitin gene. An expression vector that produces a fusion protein comprising an ubiquitin and an antigen-determining site protein of HCV envelope 1 or envelope 2 protein upon expression thereof, and the antigenic determination of envelope 1 or envelope 2 protein of HCV using the expression vector. A method for mass production of antigenic proteins comprising a site.

Description

Identification of antigenic determinants of envelope 1 and envelope 2 proteins of hepatitis C virus

FIG. 1 shows the locations of primers for amplifying the envelope gene fragments by polymerase chain reaction.

2 shows expression vectors of the envelope 1 and envelope 2 gene segments of hepatitis C virus,

Figure 3 (a) shows the results of confirming the expression of the envelope 1 and envelope 2 gene segments by SDS polyacrylamide gel electrophoresis,

Figure 3 (b) shows the results of Western blotting the expression of the envelope 1 and envelope 2 gene segments with serum of type C mesenchymal patients.

Figure 4 shows the nucleotide and amino acid sequence of the carboxyl terminus (HCV base sequence No. 1201 to 1509) of the envelope 1 protein of the Korean type hepatitis C virus (indicated by the standard single abbreviation).

FIG. 5 shows the nucleotide sequence and amino acid sequence (expressed by standard single abbreviation) at the amino terminus (HCV nucleotides 1510 to 1749) of the envelope 2 protein of the Korean hepatitis C virus.

Figure 6 shows the base sequence and amino acid sequence (indicated by standard single abbreviation) of the carboxyl terminus (HCV sequences 2281-2529) of the envelope 2 protein of the Korean hepatitis C virus.

The present invention relates to antigenic determinants of envelope 1 and envelope 2 proteins of hepatitis C virus (hereinafter referred to as "HCV").

More specifically, provided by identifying the primary immunodominant epitope that specifically reacts with the sera of hepatitis C patients using envelope 1 and envelope 2 protein segments expressed from the envelope 1 and envelope 2 genes of HCV, The present invention relates to an antigenic protein having a short length and comprising an antigenic determinant site that specifically reacts with the serum of a hepatitis C patient. Also, the genes of the antigenic determinants of the envelope 1 and envelope 2 proteins of HCV are combined with the ubiquitin gene. An expression vector producing an fusion protein comprising ubiquitin and an antigen-determining site protein of HCV envelope 1 and envelope 2 proteins at the time of its expression, and determining the antigen of the envelope 1 or envelope 2 protein of HCV using the expression vector. A method for mass production of antigenic proteins comprising a site.

Until recently, viral hepatitis has been known to be caused by hepatitis A virus (HAV), hepatitis B virus (HBV), delta hepatitis virus (HDV), cytomegalovirus (CMV) and epsinbar virus (EBV). In contrast to the above hepatitis, non-AB hepatitis B virus (NANBH) virus, which accounts for 90% of hepatitis developed after blood transfusion, has been identified (Alter, HJ, et al., Lancet 2, 838-841 (1975)). It is estimated that about 1 million people worldwide are infected with the non-A hepatitis B virus each year (Alter, HJ, AJZuckerman (ed.), Viral Hepatitis and Liver Disease, 537-542, Alan R. Liss , New York (1988)), of which about 40-50% develop chronic hepatitis and about 20% develop liver cirrhosis and liver cancer (Dienstag, JL, et al., Seminar Liver). Dis. 6, 67-81 (1986)).

Bradley et al. Reported that non-A-B virus (hereinafter referred to as HCV) can be isolated and recovered by infecting plasma from NANBH patients with chimpanzees (Bradley, DW et al., Gastroenterology, 88, 773-). 779 (1985)), Cho et al., Is a positive-stranded RNA virus that consists of about 10,000 bases and encodes a polyprotein precursor consisting of about 3010-3011 amino acids. It has been reported to have an open reading frame (ORF) (Choo, QL, et al., Science 244, 359-362 (1989); Choo, QL, et al., Proc. Natl. Acad. Sci. USA 88, 2451-2455 (1991).

The genetic structure of hepatitis C virus has similarities to those of flavivirus and pestivirus, and due to their flexibility, the polyprotein of hepatitis C virus can be transferred from the amino terminus to the nucleus. core-envelope 1-envelope 2 / unstructured 1 (E2 / NS1) -unstructured 2 (NS2) -unstructured 3 (NS3) -unstructured 4 (NS4) -unstructured 5 (NS5) (Choo, QL, et al., Proc. Natl. Aca. Sci. USA 88, 2451-2455 (1991); Takamizawa, A., et al., J. Virol. 65, 1105-1113 (1991); Kato, N., et al., Proc. Natl. Acad. Sci USA 87, 6524-6528 (1990)).

Envelope 1 and envelope 2 / unstructured 1 proteins (hereinafter referred to as envelope 2 proteins) have been found in vitro to be glycoproteins with molecular weights of 33,000 and 72,000 Daltons (Houghton, M., et al., Hepatology 14, 381-388 (1991); Hijkata, M., et al., Proc. Natl. Acad. Sci. USA 88, 5547-5551 (1991)), presumably vaccines of flaviviruses The gp53 / 55 protein, which is believed to be a vaccine of non-structural 1 protein and pestivirus, and its considerable structural history, have been the primary targets for the development of jacksin and therapies for hepatitis C virus (Spete, RR et al., Virology 188). , 819-830 (1992).

Nonstructural 3 protein is a viral protease that is supposed to generate nonstructural proteins by cutting nonstructural proteins encoded by nonstructural genes (Bajan, JF, Virology 171, 637-639 (1989)). The nonstructural 5 protein is very similar to the amino acid sequence of viral ribonucleic acid polymerase so far known, and is assumed to be the ribonucleic acid polymerase of hepatitis C virus (Mita, E., et al., Biochem. Res.Comm. 183, 925-930 (1992).

The diagnostic method for the infection of hepatitis C virus was initially fused by two nonstructural gene fragments (NS3 / NS4) with the superoxide dismutase (SOD) gene of Chiron, USA. By expressing C100-3 and using it, enzyme immunoassay demonstrated that at least 70% of transfusion hepatitis patients had antibodies to this antigen (Kuo, G., et al., Science 244, 362-384 ( 1989). However, in many patients, antibodies against C100-3 are produced 4 to 6 months after infection with hepatitis C virus, making it impossible to make an accurate diagnosis in early acute hepatitis patients (Alber, HJ et al., N Engl. J. Med 321, 1494-1500 (1989); Miyamura, T. et al., Proc. Natl. Acad. Sci. USA 87, 983-987 (1990)) and autoimmune diseases other than hepatitis C. It has been reported that there is a disadvantage in that hepatitis hepatitis due to the positive response (Mc Fararlane, IG, et al., Lancet 335, 754-757 (1990)).

Recently, a diagnostic method using a nuclear protein expressed from a core structural gene located at the 5 ′ end of hepatitis C virus can diagnose the antibody about 6 to 8 weeks earlier than the diagnostic method using C100-3. As a result, it was reported that early diagnosis is possible and sensitive diagnosis method compared to the first generation diagnosis method using only C100-3 antigen (Harada, S. et al., J. Virol. 65, 3015 (1991)). UBI of the United States reported an improved diagnostic method with higher specificity using synthetic polypeptides consisting of 15 to 65 amino acids from nuclear structural genes (Wang, CY, EP 442394 (1991)). A second-generation diagnostic method in which the antigen-binding C22 derived from the nuclear structural gene and the C33C derived from the nonstructural 3 gene was added to the antigen was reported to further increase the sensitivity and specificity for HCV antibodies (Van der poel, CL, et al. Lacet 337, 317-319 (1991).

The present inventors have reported a method for preparing an envelope protein that can be usefully used for the diagnosis of hepatitis C and the development of a vaccine, unlike other HCV specific antigens that have been used for the diagnosis of hepatitis C. See Korean Patent Application No. 92-10039).

In these antigenic proteins, antigen-determination epitopes of HCV, to which antibodies present in the serum of hepatitis C patients specifically bind, are used in the development of hepatitis C diagnostic reagents and in the immune response studies of hepatitis C patients. The preparation of antigenic proteins, which are important markers and have immunological specificities with short lengths, allows for the efficient and economical diagnosis of hepatitis and the development of vaccines. .

In the case of the C100-3 antigen, antigen-determining sites that specifically bind to the antibody are present within HCV sequences 1690 to 1731 by measuring the response of the hepatitis C patient to serum using a plurality of short synthetic peptides. (Bahl, C., et al. P65-66, 3rd International HCV Symposium, Strasbourg, France, 1991), and in the case of nuclear proteins, fragments of three different regions of the entire gene are expressed in E. coli. By determining the response of the hepatitis C patient to the serum, the antigenic determinants were located within the 222 base pairs encoding the amino terminus (Nasoft, MS et al., Proc. Natl. Acad Sci. USA 88). , 5462-5466 (1991).

When the inventors of the present invention intend to identify the antigenic determinants of the envelope 1 or envelope 2 proteins of HCV, the inventors can confirm the antigenic determinants by measuring the antigenicity of each fragment protein after preparing fragments of each coat protein with ubiquitin. It was found.

Ubiquitin is a protein with a molecular weight of 5,500 Daltons found throughout eukaryotic cells and without its genes in prokaryotic cells (Goldstein, et al., Proc. Natl. Acad. Sci. 72,11 (1975)). The ubiquitin found in the cells was found to be the same, and there are many important roles of ubiquitin, but among them, proteolysis is known to be important (Finley, et al., Trends Biochem. Sci. 10,343 (1985)). In Escherichia coli, the lack of the ubiquitin system results in the accumulation of the fusion protein as it is in the cell without degradation of the gene product expressed as a ubiquitin fusion protein in the cell. This fusion protein system is particularly useful because the ubiquitin fused to protect the protein to be easily degraded by the protease in the cell, and can be confirmed by using an anti-ubiquitin antibody It can also be usefully used for purification.

Recently, ubiquitin cleavage enzyme named UBP 1 has been isolated and identified in yeast to obtain only the target protein from which ubiquitin has been removed from E. coli-derived ubiquitin fusion protein (Tobias, JW et al., J. Biological Chemistry 266, 12021). -12028 (1991).

Based on this fact, the present inventors fused the envelope 1 or envelope 2 gene fragment of HCV with the ubiquitin gene to express it as a fusion protein in E. coli, and then measured the Western-blotting immunoreactivity with hepatitis C patients. The present invention has been completed by finding that the antigenic sites are concentrated at the carboxyl terminus of the coat 1 protein, the amino terminus and the carboxyl terminus of the coat 2 protein.

In other words, it is an object of the present invention to provide antigenic proteins that maintain the sensitivity and accuracy while having the shortest possible length by identifying antigenic sites of envelope 1 and envelope 2 proteins of hepatitis C virus. The present invention provides a recombinant antigenic protein fused to ubiquitin, comprising the antigenic determining site of the envelope 1 or envelope 2 protein.

Another object of the present invention is to determine the antigen of the envelope 1 or envelope 2 protein of HCV in its expression, including the gene region encoding the antigen-determination site protein of the envelope 1 or envelope 2 protein of the HCV in combination with the ubiquitin gene An expression vector capable of producing a fusion ene protein containing a site and ubiquitin, a microorganism transformed with the expression vector, and an antigenic protein including an antigen-determining site of the envelope 1 or envelope 2 protein of HCV using these microorganisms. It is to provide a way to produce.

Here, the envelope protein is meant to include the envelope 1 and envelope 2 proteins. The envelope protein fragment herein refers to a protein in which one or more amino acids are deleted from the envelope 1 or envelope 2 protein.

Hereinafter, the present invention will be described in detail.

First, the envelope 1 or envelope 2 gene encoding each envelope 1 or envelope 2 protein fragments from sequencing information on cDNA fragments of the Korean hepatitis C virus (see Korean Patent Application No. 92-10039 previously filed by the applicant). Primers for polymerase chain reaction (PCR) corresponding to the 5 ′ and 3 ′ ends of the fragments were synthesized. The primer corresponding to the 5 'end of the gene fragment has a restriction enzyme recognition site to enable conjugation with the 3' end of the ubiquitin gene at the 5 'end, and the primer corresponding to the 3' end has the end codon and the restriction enzyme. By inserting the recognition site, the protein is synthesized from the start codon of ubiquitin to complete the protein synthesis at the end codon inserted artificially and to facilitate cloning into the expression vector.

Using these primers, the envelope 1 gene (ATCC 68878, deposited on Dec. 11, 1991) and the envelope 2 gene (ATCC 69866 and ATCC 74117, deposited on Dec. 11, 1991) were used as a template. And amplifying the envelope 2 gene fragments, and each of the gene fragments obtained by cleaving the amplified genes with restriction enzymes was plasmid ptrpH-UB-KHCV (ATCC 68640, ATCC 68641, ATCC 68642) (Korean Patent Application No. Each expression vector was prepared by substituting the KHCV gene (see No. 92-10039). Each envelope 1 and envelope 2 protein fragments expressed from the above expression vector were electrophoresed on 15% polyacrylamide gel, and then Western-blotted with immunoglobulins isolated from the serum of Korean hepatitis C patients. Protein fragments that specifically react to the antibodies of the virus were identified and their positions within the HCV gene.

That is, in the case of the envelope protein of HCV, the main antigenic determinant is the carboxyl terminus of the envelope 1 protein expressed from 309 base pairs corresponding to HCV sequences 1201 to 1509 (FIG. 4); Amino terminus of envelope 2 protein expressed from 240 base pairs corresponding to HCV sequences 1510-1749 (FIG. 5); And the carboxyl terminus (FIG. 6) of the envelope 2 protein expressed from 249 base pairs corresponding to HCV sequences 2281-2529.

Diagnosis of hepatitis C is more economical, accurate, and more sensitive than antibody diagnostic methods using proteins obtained from the entire envelope 1 and envelope 2 genes by using envelope protein fragments concentrated in the major antigenic determinants identified by the present invention. This became possible.

Hereinafter, the present invention will be described in more detail with reference to Examples. The following examples merely illustrate the invention and do not limit the scope of the invention. Experimental methods and procedures used in the following examples were carried out according to the following Reference Examples 1) -6) unless otherwise specified.

Reference Example 1

DNA cleavage using restriction enzymes

Restriction enzyme and reaction buffer were added to a sterile 1.5 ml eppendorf tube to a reaction volume of 50 µl to 100 µl and reacted at 37 ° C. for 1 to 2 hours. After the reaction was completed, heat treatment was performed at 65 ° C. for 15 minutes to inactivate the restriction enzyme, and phenol extraction and ethanol precipitation were used for the restriction enzyme which was heat resistant. Restriction enzymes and reaction buffers used were purchased from New England Biolabs, MA, USA. The composition of the 10-fold reaction buffer is as follows.

10-fold NEB buffer 1: 100 mM bis tris propane-HCl, 100 mM MgCl ₂ , 10 mM dithiothreitol (DTT), pH 7.0

10-fold NEB buffer 2: 100 mM Tris-HCl, 100 mM MgCl ₂ , 500 mM NaCl, 10 mM DTT, pH 7.0

10-fold NEB buffer 3: 100 mM Tris-HCl, 100 mM MgCl ₂ , 1000 mM NaCl, 10 mM DTT, pH 7.0

10-fold NEB buffer 4: 200 mM tris-acetate, 100 mM magnesium acetate, 500 mM potassium acetate, 10 mM DTT, pH 7.0

Reference Example 2

[Phenol Extraction and Ethanol Precipitation]

Phenol extraction was used to deactivate the restriction enzyme or extract the nucleic acid after the restriction enzyme reaction was completed. Phenol was used after equilibration with 10 mM Tris-HCl (pH 8.0), 1 mM EDTA solution.

The sample and phenol are mixed at a ratio of 1: 1 (v / v), shaken vigorously, and then centrifuged (15,000xG, 5 minutes) to transfer the supernatant to a new tube. After repeating the same process for 3 times, extracting the supernatant with the same volume of chloroform solution (chloroform to isobutanol ratio 24: 1 (v / v)), 0.1 volume of 3M sodium acetate and 2.5 volume of 100% ethanol Add. This was allowed to stand at -70 ° C for 30 minutes or at -20 ° C for at least 12 hours, followed by centrifugation (15,000xG, 20 minutes, 4 ° C) to precipitate the nucleic acid.

Reference Example 3

[Ligation]

T4 DNA ligase (T4 DNA ligase, New England Biolabs, Inc.) and 10-fold ligation buffer (0.5M Tris-HCl, pH7.8, 0.1M MgCl ₂ , 0.2M DTT, 10mM ATP, 0.5mg) as ligase Ligation was performed using / mL BSA (bovine serum albumin)). Usually, 10 units of ligating enzyme is used in a volume of 20 μl, and 100 units of ligating enzyme is used for ligation of DNA having blunt ends. At least 5 hours at 16 ° C. or 14 hours at 4 ° C. After completion of the reaction, the ligase was inactivated by heating at 65 ° C. for 15 minutes.

Reference Example 4

[E. coli transformation]

When the E. coli host cells (E. coli HB101, W3110 or JM105) were inoculated into 100 ml of liquid LB medium (1% bactotrypton, 0.5% yeast extract, 1% sodium chloride), the absorbance value at 650 nm reached 0.25 to 0.5. Shake culture at 37 ℃ until. Cells are then separated by centrifugation (2,500 × G, 10 minutes) and then washed by adding 50 ml of 0.1 M MgCl ₂ . It was centrifuged again (2,500 × G, 10 minutes) and 50 ml of 0.1M CaCl ₂ , 0.05M MgCl ₂ solution was added thereto and allowed to stand on ice for 30 minutes. This was again centrifuged (2,500 xG, 10 minutes) and uniformly dispersed in 5 ml of the same solution (0.1M CaCl ₂ , 0.05M MgCl ₂ ). All solutions and vessels used above were used after cooling at 0 ° C. 10 μl of the ligation reaction solution was added to 0.2 ml of the cell suspension obtained above, and the mixture was left at 0 ° C. for 40 minutes and then heat-treated at 42 ° C. for 1 minute. This was added to 2.0 ml of liquid LB medium and incubated at 37 ° C. for 1 hour, and the culture solution was plated on solid LB medium containing 50 μg / ml of ampicillin, and then cultured overnight at 37 ° C. to form colonies. It was. M13 vector DNA was heat treated and then 100 μl of E. coli JM105 cells, 15 μl of 100 mM IPTG (isopropyl-β-D-thiogalactoside), 25 μl of 4% X-Gal (5-bromo-4-chloro-indolyl- β-D-galactoside) and 3 ml of 2-fold soft solid YT medium (1% NaCl, 1% yeast extract, 1.6% bacto tryptone, 8.7% bacto agar) pre-warmed to 48 ° C. Plated on pear solid YT medium (1% NaCl, 1% yeast extract, 1.6% bacto tryptone, 1.5% bactoagar).

Reference Example 5

[Synthesis of oligomers]

The oligomers were synthesized by a DNA synthesizer (Applied Biosystems, Inc., model 380 B, USA) using automated solid phase phosphoamidite chemistry and then modified polyacrylamide gel (2M urea, 12% acrylamide). And electrophoresis using bis (29: 1 w / w), 50 mM boric acid, 1 mM EDTA) in 50 mM Tris. Next, it was attached to SEP-PAK (waters Inc., USA), a C ₁₈ column, and purified using pure acetonitrile: water mixture (50:50 (v / v)) as eluent, and absorbance was measured at 260 nm. The concentration was determined by.

Reference Example 6

[Polymerase Chain Reaction (PCR)]

Template DNA (10 ng to 100 ng), 10 μg, 10-fold concentration of Taq polymerase buffer solution (10 mM Tris-Cl pH 8.3, 500 mM KCl, 15 mM MgCl ₂ , 0.1% (w / v) gelatin), 10 μl of dNTP mixed solution (1.25 mM each for dGTP, dATP, dTTP, and dCTP), 2 μg of primer (usually 2 primers were used, and 20ng was used for intermediate primers when using three primers simultaneously), 0.5 μl AmpliTaq DNA polymerization Distilled water was added to the enzyme (Perkin Elmer Cetus, USA) to make a total volume of 100 μl. 50 μl of mineral oil was added to prevent evaporation of the solution. A temperature cycler (Perkin Elmer Cetus, USA) was used, and the cycle program was repeated 25 times for 1 minute at 95 ° C, 1 minute at 55 ° C, and 2 minutes at 72 ° C and finally further reacted at 72 ° C for 10 minutes. .

In order to remove the polymerase after the reaction, the reaction mixture was added with phenol / cooloform of eastern blood and mixed well, followed by centrifugation. The supernatant was transferred to a new tube, mixed with 1/10 volume of 3M sodium acetate and 2 volumes of 100% ethanol, followed by centrifugation to obtain a double helix nucleic acid. The nucleic acid was dissolved in 20 μl of TE buffer (10 mM Tris-Cl, pH 7.5, 1 mM EDTA) and used for the next experiment.

Example 1

[Amplification of Cortex 1 and Envelope 2 Gene Segments of HCV]

(Step 1) Synthesis of the primer

PCR primers were synthesized as follows to obtain respective gene segments of envelope 1 and envelope 2 of hepatitis C virus.

Primer PE1T2 (5′-TGAGACTCCGCGGTGGTTATGAAGTGGGCAACGCGTCC-3 ′)

(It has restriction enzyme SacII recognition site and contains 1st to 21st base of coat 1 gene)

Primer PE1DT2 (5′-TGAGACTCCGCGGTGGTGACTTGCTCGTTGGGGTAGCT-3 ′)

(It has restriction enzyme SacII recognition site and contains 214th to 234th base of coat 1 gene)

Primer PE1EGT2 (5′-TGAGACTCCGCGGTGGTGTTTCCCAGCTGTTCACCTTC-3 ′)

(It has restriction enzyme SacII recognition site and contains 286th to 306th base of coat 1 gene)

Primer PE1FT2 (5′-TGAGACTCCGCGGTGGTACAACAGCCCTAGTGGTATCG-3 ′)

(It has restriction enzyme SacII recognition site and contains 412th base to 432th base of coat 1 gene)

Primer PE1AXH0 (5′-AAAAAACTCGAGTTAGACATGGCGTCGCAATGTCGT-3 ′)

(It has an end codon that terminates translation after the 213th base of coat 1 gene and at the same time has a restriction enzyme Xhol recognition site)

Primer PE1BXH0 (5′-AAAAAACTCGAGTTAAAGGAAAACAGATCCGCAGAG-3 ′)

(With end codon to terminate translation after 285th base of coat 1 gene and at the same time has restriction enzyme Xhol recognition site)

Primer PE1CDEXH0 (5′-AAAAAACTCGAGTTAAGGCGACCAGTTCATCATCAT-3 ′)

(It has an end codon which terminates translation after the 411th base of coat 1 gene and at the same time has a restriction enzyme Xhol recognition site)

Primer PE1XH0 (5′-AAAAAACTCGAGTTACCCTGTCACGTGGGTGGTTCC-3 ′)

(With end codon to terminate translation after 594th base of coat 1 gene and at the same time has restriction enzyme Xhol recognition site)

Primer PE2T2 (5′-TGAGACTCCGCGGTGGTGGGGCGCAAGGTCGGGCCGCT-3 ′)

(It has restriction enzyme SacII recognition site and contains 1st to 21st bases of coat 2 gene)

Primer PE2T2 (5′-TGAGACTCCGCGGTGGTGGTCCCATCACTTACACTGAG-3 ′)

(It has restriction enzyme SacII recognition site and contains 241st base to 261th base of envelope 2 gene)

Primer PE2DFT2 (5′-TGAGACTCCGCGGTGGTGGCACTGGGTTCACCAAGACA-3 ′)

(It has restriction enzyme SacII recognition site and contains 502th base to 522th base of envelope 2 gene)

Primer PE2ET2 (5′-TGAGACTCCGCGGTGGTACTCGGGGAGAGCGTTGTGAC-3 ′)

(It has restriction enzyme SacII recognition site and contains 772th base to 792th base of envelope 2 gene)

Primer PE2AXH0 (5′-AAAAAACTCGAGTTACCACCCCTGCGCGAATGTATC-3 ′)

(With end codon to terminate translation after 240th base of envelope 2 gene and at the same time has restriction enzyme Xhol recognition site)

Primer PE2BCXH0 (5′-AAAAAACTCGAGTTAATTCATCCAGGTACAACCGAA-3 ′)

(It has an end codon that terminates translation after the 501st base of envelope 2 gene and at the same time has a restriction enzyme Xhol recognition site)

Primer PE2DXH0 (5′-AAAAAACTCGAGTTACCAGTTGCATGCGGCGTCGAG-3 ′)

(It has an end codon that terminates detoxification after the 771th base of the envelope 2 gene and at the same time has a restriction enzyme Xhol recognition site)

Primer PE2XH0 (5′-AAAAAACTCGAGTTACGCGTCCGCCAGAAGAAGGAAGAG-3 ′)

(With end codon to terminate translation after 1020th base of envelope 2 gene and at the same time has restriction enzyme Xhol recognition site)

(Step 2) polymerase chain reaction (PCR)

2µg of primer PE1T2 and 2µg of primer PE1XH0 in reaction tube A

2µg of primer PE1T2 and 2µg of primer PE1AXH0 in reaction tube B

2µg of primer PE1T2 and 2µg of primer PE1BXH0 in reaction tube C

2µg of primer PE1T2 and 2µg of primer PE1CDEXH0 in reaction tube D

2µg of primer PE1DT2 and 2µg of primer PE1CDEXH0 in reaction tube E

2µg of primer PE1EGT2 and 2µg of primer PE1CDEXH0 in reaction tube F

2µg of primer PE1FT2 and 2µg of primer PE1XH0 in reaction tube G

2µg of primer PE1EGT2 and 2µg of primer PE1XH0 in reaction tube H

2µg of primer PE2T2 and 2µg of primer PE2AXH0 in reaction tube I

2µg of primer PE2BT2 and 2µg of primer PE2BCXH0 in reaction tube J

2µg of primer PE2T2 and 2µg of primer PE2BCXH0 in reaction tube K

2µg of primer PE2DFT2 and 2µg of primer PE2DXH0 in reaction tube L

2µg of primer PE2ET2 and 2µg of primer PE2XH0 in reaction tube M

2µg of primer PE2DFT2 and 2µg of primer PE2XH0 in reaction tube N

Put each of them into the A-H tube as a template, the outer shell 1 gene, the I to N tube 50 gene each of the outer shell 2, 10μl 10-fold polymerization buffer solution, 10μl 2mM dNTP (2mM dGTP, 2mM dATP, 2 mM dTTP, 2 mM dCTP), 2.5 μl of 10-unit Taq polymerase and distilled water were added to make the total volume 100 μl, then 30 seconds at 95 ° C., 30 seconds at 55 ° C., 72 ° C. as in Reference Example 6. A 25 minute polymerase chain reaction was performed (Saiki, RK et al., Science 239, 487 (1988)).

(Step 3) Isolation and Purification of PCR Products

Each PCR product obtained in step 2 was separated from a 5% polyacrylamide gel. As a result, about 600 base pairs of DNA in reaction tube A, about 220 base pairs of DNA in tube B, and about 300 base pairs of DNA in tube C, tube D. Is about 410 base pairs of DNA, about 200 base pairs of DNA in tube E, about 110 base pairs of DNA in tube F, about 190 base pairs of DNA in tube G, about 300 base pairs of DNA in tube H, and about 210 base pairs in tube I. DNA, about 300 base pairs of DNA in tube J, about 505 base pairs of DNA in tube K, about 290 base pairs of DNA in tube L, about 240 base pairs of DNA in tube M, and about 520 base pairs of DNA in tube N It was confirmed that they were separated and purified by polyacryl amide gel of the same conditions. Hereinafter, each DNA fragment is named Fragment E1, Fragment E1A, Fragment E1B, Fragment E1C, Fragment E1D, Fragment E1E, Fragment E1F, Fragment E1G, Fragment E2A, Fragment E2B, Fragment E2C, Fragment E2D, Fragment E2E and Fragment E2F. It was.

Example 2

[Production of Escherichia Coli Expression Vector]

2 μg of plasmid ptrpH-UB-CORE14 (Korean Patent Application No. 92-10039, ATCC 68642, dated Jul. 1, 1991) filed by the Applicant of NEB buffer 3 with restriction enzyme SacII and restriction enzyme SaII After complete cleavage under the conditions, the 2.7 Kb fragment was separated and purified by separation on a 0.7% agarose gel.

This fragment is hereinafter referred to as ptrpH-UB-T2 / L.

Meanwhile, 2 μg of each DNA fragment obtained in step 3 of Example 1 was completely cut with restriction enzyme ScaII and restriction enzyme Xhol under the conditions of NEB buffer 3 as in Reference Example 1, and then 100 ng DNA fragments cut into each tube. , 50ng fragment ptrpH-UB-T2 / L, 2µl of 10-fold ligation solution, 10 units of T4 DNA ligase was added and distilled water was added so that the total volume was 20µl and reacted at 16 ° C for 12 hours. . After the reaction, E. coli W3110 (ATCC 37339) was used to transform ptrpH-UBE1 containing fragment E1, ptrpH-UBE1 containing fragment E1A, ptrpH-UBE1B containing fragment E1B, and fragment E1C. One ptrpH-UBE1C, ptrpH-UBE1D with fragment E1D, ptrpH-UBE1E with fragment E1E, ptrpH-UBE1F with fragment E1F, ptrpH-UBE1G with fragment E1G, ptrpH-UBE2A with fragment E2A, PtrpH-UBE2B with fragment E2B, ptrpH-UBE2C with fragment E2C, ptrpH-UBE2D with fragment E2D, ptrpH-UBE2E with fragment E2E and ptrpH-UBE2F with fragment E2F were obtained, respectively.

Example 3

[Induction of Expression of Envelope 1 and Envelope 2 Gene Segments]

Recombinant Escherichia coli cells containing envelope 1 or envelope 2 gene segments of hepatitis C virus obtained in Example 2 were prepared in liquid Luria medium (6% bactotrypton, 0.5%) containing 10 μg / ml of ampicillin. After shaking for 12 hours in yeast extract, 1% sodium chloride, 3 ml each of 300 ml of M9 medium (400 mM K ₂ HPO ₄ , 22 mM KH ₂ PO ₄ , 8.5 mM NaCl, 18.7 mM NH ₄ Cl, 1) was added. % Glucose, 0.1 mM MgSO ₄ , 0.1 mM CaCl ₂ , 0.4% casamino acid, 10 μg / ml Vit.B ₁ , 40 μg / ml ampicillin) and incubated at 37 ° C. for about 4 hours to absorb absorbance of the culture at 650 nm. When the wavelength was about 0.3, indole acrylic acid (IAA) was added to a final concentration of 50 µg / ml. About 4 hours after the addition of IAA, the absorbance of each cell culture was measured and centrifuged (Beckman J2-21, JA14 rotor) for 25 minutes at 11,000rpm to collect E. coli cell precipitate.

Example 4

[Identification of Immundominant Epitope]

The cell precipitates of Example 3 were electrophoresed on 15% polyacrylamide gels in the presence of SDS according to the method of Laemmli, Nature 227, 680 (1970), and the proteins isolated on the gels were isolated from Tovin's method (Towvin). , et al., Proc. Natl. Acad. Sci. USA 76, 4750 (1979)), were transferred to a nitro cellulose filter (Bio-Rad Lab., pore size 0.22 μm, CA, USA). The filter was placed in PBS (10 mM phosphoric acid, pH 7.0, 0.15 M sodium chloride) containing 0.5% tween 20 and closed with gentle shaking for 2 hours at room temperature. Afterwards, immunoglobulin (IgG) isolated from the serum of Korean hepatitis C patients was added to PBS containing 0.5% gelatin and 0.05% Tween 20 to a final concentration of 16 µg / ml and gently shaken at room temperature for 1 hour. After the reaction, the filter was washed four times for 5 minutes with PBS containing 0.2% Tween 20. The filter was labeled with anti-human immunoglobulin G (Bio-Rad Lab, anti-human IgG-HRP, 0.01 mg.ml, labeled with PBS horseradish peroxidase containing 0.5% gelatin and 0.05% Tween 20. CA, USA) was added at a dilution of 1: 500 and reacted by shaking for 1 hour at room temperature, washed 4 times with PBS containing 0.2% Tween 20 for 5 minutes, and then washed twice with 50 mM Tris buffer (pH 7.0). It was. The filter was developed by adding 50 mM Tris buffer containing 400 µg / ml 4-chloro-1-naphthol and 0.03% hydrogen peroxide water. The results are shown in FIG.

In FIG. 3, the first column is E. coli without plasmid, the second column is E. coli containing ptrpH-UBE1, the third column is E. coli containing ptrpH-UBE1A, and the fourth column is E. coli containing ptrpH-UBE1B. Column 5 is E. coli containing ptrpH-UBE1C, column 6 is E. coli containing ptrpH-UBE1D, column 7 is E. coli containing ptrpH-UBE1E, column 8 is E. coli containing ptrpH-UBE1F, and Column 9 is Escherichia coli containing ptrpH-UBE1G, Column 10 is Escherichia coli containing ptrpH-UBE2A, Column 11 is Escherichia coli containing ptrpH-UBE2B, Column 12 is Escherichia coli containing ptrpH-UBE2C, Column 13 Is E. coli containing ptrpH-UBE2D, column 14 shows E. coli containing ptrpH-UBE2E, and column 15 shows cell precipitates obtained from E. coli containing ptrpH-UBE2F.

The results of Western blotting confirming reactivity with immunoglobulins isolated from serum of hepatitis C patients using cell precipitates of Escherichia coli containing envelope 1 and envelope 2 protein fragments are shown in Table 1 and FIG. 3B. .

TABLE 1

As can be seen from Table 1 and FIG. 3B, fragment E1G of the carboxyl terminus of the coat 1 protein, fragment E2A of the amino terminus of the coat 2 protein and carboxyl in Western blotting using serum of Korean hepatitis C patients In the case of the envelope protein of HCV, the major antigenic determinant site was HCV nucleotide 1201, since the fragment E2E, which is terminal, was positive, but E1A, E1B, E1C, E2B, E2D, etc., including other gene parts, were negative. Carboxyl terminus of coat 1 protein expressed from 309 base pairs corresponding to steps 1 to 1509 (FIG. 4); Amino terminus of envelope 2 protein expressed from 240 base pairs corresponding to HCV sequences 1510-1749 (FIG. 5); And HCV nucleotides 2281 to 2529 were found at the carboxyl terminus (Fig. 6) of the envelope 2 protein expressed from 249 base pairs.

Claims (16)

A fragment of the envelope protein that maintains the immunological specificity of the Korean hepatitis C virus envelope protein, or a recombinant protein comprising the same. The recombinant protein of claim 1, wherein the carboxyl terminal protein fragment of the coat 1 protein of the Korean hepatitis C virus having the following amino acid sequence, or the same. The recombinant protein of claim 1, wherein the amino terminal protein fragment of the envelope 2 protein of the Korean hepatitis C virus having the following amino acid sequence, or the same. The recombinant protein of claim 1, wherein the carboxyl terminal protein fragment of the envelope 2 protein of the Korean hepatitis C virus having the following amino acid sequence, or the same. The recombinant protein according to any one of claims 1 to 4, wherein ubiquitin is fused to the coat protein fragment. A DNA having a nucleotide sequence encoding the protein of any one of claims 1 to 5. 7. The DNA of claim 6 comprising the following nucleotide sequence encoding the carboxyl terminal protein fragment of coat 1 protein of the Korean hepatitis C virus. The DNA of claim 6 comprising the following nucleotide sequence encoding the amino terminal protein fragment of the envelope 2 protein of the Korean hepatitis C virus. 7. The DNA of claim 6 comprising the following nucleotide sequence encoding the carboxy terminal protein fragment of the envelope 2 protein of the Korean hepatitis C virus. The recombinant DNA according to any one of claims 6 to 9, wherein a ubiquitin gene is fused to a nucleotide sequence encoding the coat protein. An expression vector comprising the DNA of any one of claims 6 to 10. The expression vector of claim 11 which is ptrpH-UBE1G, ptrpH-UBE2A or ptrpH-UBE2E. Escherichia coli transformed with the expression vector of claim 11 or 12. The E. coli transformed with ptrpH-UBE1G, ptrpH-UBE2A or ptrpH-UBE2E. A method for producing a protein fragment or a recombinant protein comprising the same, the method comprising maintaining the immunological specificity of the envelope protein of the Korean hepatitis C virus, the method comprising culturing Escherichia coli according to claim 13 or 14. 16. The method of claim 15, comprising culturing Escherichia coli transformed with ptrpH-UBE1G, ptrpH-UBE2A, or ptrpH-UBE2E.