KR100301795B1 - Nucleotide sequence and amino acid sequence of hcv hypervariable region - Google Patents

Abstract

PURPOSE: Provided are a nucleotide sequence of HCV hypervariable region and an amino acid sequence derived therefrom. CONSTITUTION: An HCV polypeptide contains at least one of displaced amino acid in HCV hypervariable region of 384-410 amino acids, represented by TTHTTGGTAARTTSGLTSFSPGPAQK. The polypeptide can be manufactured by the steps of: preparing an expression vector including DNA which contains a nucleotide sequence of HCV polypeptide; transforming a microbe with the vector; and culturing the transformed microbe.

Description

Nucleotide and Amino Acid Sequences of the Hypervariable Regions of Korean Hepatitis C Virus

Figure 1 shows the nucleotide sequence of fragment HV-A and the amino acid sequence inferred therefrom,

Figure 2 shows the nucleotide sequence of fragment HV-A1 and the amino acid sequence inferred therefrom,

Figure 3 shows the nucleotide sequence of fragment HV-B and the amino acid sequence inferred therefrom,

Figure 4 shows the nucleotide sequence of fragment HV-23 and the amino acid sequence inferred therefrom,

Figure 5 shows the nucleotide sequence of fragment HV-6 and the amino acid sequence inferred therefrom,

6 shows the nucleotide sequence of fragment HV-27B and the amino acid sequence inferred therefrom,

Figure 7 shows the nucleotide sequence of fragment HV-H1 and the amino acid sequence inferred therefrom,

Figure 8 shows the nucleotide sequence of fragment HV-H4 and the amino acid sequence inferred therefrom,

Figure 9 shows the nucleotide sequence of fragment HV-H5 and the amino acid sequence inferred therefrom,

Figure 10 shows the nucleotide sequence of fragment HV-H6 and the amino acid sequence inferred therefrom,

Figure 11 shows the nucleotide sequence of fragment HV-H12 and the amino acid sequence inferred therefrom,

Figure 12 shows the nucleotide sequence of fragment HV-H19 and the amino acid sequence inferred therefrom,

Figure 13 shows the nucleotide sequence of fragment HV-H28 and the amino acid sequence inferred therefrom,

14 shows the nucleotide sequence of fragment HV-H34 and the amino acid sequence inferred therefrom,

Figure 15 shows the nucleotide sequence of fragment HV-HC6 and the amino acid sequence inferred therefrom,

Figure 16 shows the nucleotide sequence of fragment HV-L14 and the amino acid sequence inferred therefrom,

Figure 17 shows the nucleotide sequence of fragment HV-F1 and the amino acid sequence inferred therefrom,

18 shows the nucleotide sequence of fragment HV-F2 and the amino acid sequence inferred therefrom,

Figure 19 shows the nucleotide sequence of fragment HV-F3 and the amino acid sequence inferred therefrom,

Figure 20 shows the nucleotide sequence of fragment HV-I3 and the amino acid sequence inferred therefrom,

Figure 21 shows the nucleotide sequence of fragment HV-I20 and the amino acid sequence inferred therefrom,

22 shows a comparison of the amino acid sequences of 21 clones.

The present invention relates to a hypervariable region of NANBH virus or hepatitis C virus (HCV) obtained from a Korean patient, Non-A, Non-B Hepatitis (NANBH). Nucleotide sequences and amino acid sequences inferred therefrom.

In general, viral hepatitis includes Hepatitis A Virus (HAV), Hepatitis B Virus (HBV), Hepatitis Delta Virus (HDV), Hepatitis E Virus (HEV), and Cyto. It has been known to be caused by megalovirus (CMV) and Epsin bar virus (EBV), and its genes have been identified in the 1980s, and diagnostic reagents and vaccines have been developed using them, and thus, much progress has been made in the diagnosis and prevention of hepatitis.

However, a different form of hepatitis, called non-A, non-B hepatitis, which differs from the above, accounts for 80-90% of hepatitis after transfusion (Lancet 2, 838-841 (1975)) and about 50% of patients Is known as a terrible epidemic that spreads to cirrhosis and hepatocellular carcinoma, and it has only been found that human non-A hepatitis B can infect chimpanzees (Hollinger, FB et al., Intervirology 10 , 60-68 (1978); Bradley, DW et al., J. Med. Virol. 9, 253-269 (1979)), the nature and mechanisms of antigen-antibodies associated with non-A non-B hepatitis have recently been revealed. I didn't lose. The reason for this is that a small number of viruses are present in the blood of non-A hepatitis B patients and there is no information related to the antigens and antibodies of the virus.

Bradley et al. Infected the plasma of NANBH patients with chimpanzees, secured a large amount of hepatitis serum, and established methods for the isolation and recovery of viruses (Bradley, DW et al., Gastroenterology 88, 773-779 (1985). The physicochemical properties of the virus have been elucidated, opening the way for analysis and application at the molecular level through genetic engineering studies.

Cho et al. Obtained the NANBH virus using a large amount of chimpanzee serum obtained by the above method, extracted the whole nucleic acid of NANBH and cloned the partial cDNA (HCPT), and the gene was composed of about 10,000 base pairs. (Science 244, 359-362 (1989)), and proteins (5-1-1, C100) produced by expressing cloned partial hereditary fragments in E. coli and yeast were obtained from serum of hepatitis C patients. Reacted with (Science 244, 362-364 (1989)).

Meanwhile, the Japanese team cloned partial cDNA from the Japanese sera diagnosed with NANBH by the method described above (Kubo, Y. et al., Nucleic Acids Res. 17, 10367-10372 (1989); Kato, N. et al., Proc. Japan Acid. 65 (SerB), 219-223 (1990); Kaneko, S. et al., Lancet 335, 976 (1990); Takeuchi, K. et al., Gene 91, 287-291 (1990) Takeuchi, K. et al., Nucleic Acids Res. 18 (15), 4626 (1990); Takamixawa, A. et al., J. Virol. 65, 1105-1113 (1991)); It was claimed that the subtype was different from the nucleotide sequence of the hepatitis C virus obtained after infection.

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 is nucleus from the amino terminus. (core) -envelope 1-envelope2 / unstructured 1 (E2 / NS1) -unstructured 2 (NS2) -unstructured 3 (NS3) -unstructured 4 (NS4) -unstructured 5 (NS5) (Choo, QL, et al., Proc. Natl. Acad. 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 / nonstructural 1 proteins were 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); Hijikata, M., et al., Proc. Natl. Acad. Sci. USA 88, 5547-5551 (1991)), envelope 2 / non-structural 1 protein is a non-structural 1 ( NS1) Has significant structural similarity to gp55 (bovine viral diarrhea virus) or gp53 (pig cholera virus) of proteins and pestiviruses (Choo et al., Proc. Natl. Acad. Sci. USA 88, 2451-2455 ( 19991)).

In addition, pestivirus gp55 or gp53 and flavivirus NS1 are known to produce protective or protective antibodies when injected with these proteins, but the envelope glycoprotein of HCV is not known for this role. not.

In the case of the HIV-1 virus, antibodies to the V3 region, a hypervariable region in the envelope glycoprotein gp120, have been reported to have the ability to neutralize the virus (Gorny et al., J. of Virology 66, 7538-7542 (1992).

The E2 / NS1 protein (gp72) of HCV was also found to have a hypervariable (HV) region at the amino terminus (Kato et al., BBRC Vol. 189, 119-127 (1992)) but its properties are still unknown. I didn't lose.

Therefore, nucleotide sequence and amino acid sequence information of various variants of the hypervariable region can provide information on the mechanism of HCV infection, and will be expected to contribute significantly to vaccine development or drug development in the future.

It is an object of the present invention to provide cDNA and fragments thereof of the hypervariable region of Korean hepatitis C virus having the nucleotide sequence and amino acid sequence of FIGS.

It is another object of the present invention to provide a vector comprising the cDNA and a microorganism transformed therewith.

Another object of the present invention is to provide a polypeptide or fragment thereof encoded by the cDNA by culturing the microorganism.

Hereinafter, the present invention will be described in detail.

First, the total RNA of HCV was extracted by RNAzol B method (Chomczynski et al., Anal. Biochem. 162, 156-159 (1987)) from serum of different Korean hepatitis C patients, and then 20 units of ribohexane degrading enzyme was extracted. Dissolve in 10 μl of Diethylpyrocarbonate (DEPC) -treated distilled water containing an RNAse inhibitor (Promega, 2800 Woods Hollow Rd., Madison, W1 53711 USA).

CDNA is prepared using the cDNA synthesis kit using the RNA as a template for reverse transcriptase. At this time, the primers of the reverse transcriptase are random primers (5′-NNNNNN-3 ′, N means base G, A, T, and C are mixed in equal proportion), and the reverse transcriptase MMLV reverse transcriptase (Molony Murine). Leukemia Virus Reverse Transcriptase (MMLV-RT), and BRL's cDNA Synthesis Kit (Cat. No. 8267SA) are used as a synthesis kit.

Meanwhile, nucleotide sequence information of cDNA fragments of Korean hepatitis C virus extracted from a Korean hepatitis C patient (see Korean Patent Application No. 92-10039, filed by the applicant), and nucleotide sequence information of Japanese hepatitis C virus (Kato et al., Proc. Natl. Acad. Sci. USA 87, 9524-9528 (1990); Takamizawa et al., J. Virol. 65, 1105-1113 (1991)) and US hepatitis C virus. From the nucleotide sequence information (Choo et al., Science 244, 359-363 (1989)), the nucleotide sequence of the gene region (nucleotide sequences No. 1480 to 2529) encoding the E2 / NS1 protein (gp72) of HCV A relatively conserved portion is selected to synthesize the corresponding primer pair for the polymerase chain reaction. The primers for the polymerase chain reaction have a BamHI recognition site or SalI recognition site at the 5'-end to facilitate cloning, and a DNA synthesizer using automated solid phase phosphoamidite chemistry. (Applied Biosystems Inc., Model 380 BUSA) synthesized and electrophoresed in a modified polyacrylamide gel and purified by pure separation on C18 column SEP-PAK (Waters Inc., USA). These primers were used as a template for the cDNA of the Korean type hepatitis C virus prepared above, using a temperature circulator (Perkin-Elmer Cetus, U.S.A) at 95 ° C. for 2 minutes; The cDNA fragments are amplified by a polymerase chain reaction of 40 cycles of 55 ° C., 2 minutes, 72 ° C., and 3 minutes of temperature cycling.

Amplified cDNA fragments encoding the E2 / NS1 moiety comprising the HV region are separated by polyacrylamide gel electrophoresis and cleaved with restriction enzymes BamHi and SalI, respectively. The cleaved cDNA fragments were cloned into nucleotide sequencing vector M13mp18 (New England Biolabs, Cat. # 408) (Maniatis et al., Molecular Cloning, pp 51-53, A Laboratory Mannual, Cold Spring Harbor Laboratory (1982)). Nucleotide sequences are determined according to Sanger's dideoxy method (Sanger et al., Proc. Natl. Acad. Sci. 74, 5463 (1977)) (see FIGS. 1 to 21).

Comparing the amino acid sequences inferred from the determined nucleotide sequences shows significant changes from the 384th amino acid to the 410th amino acid (“hyperchange region”), based on the total amino acid sequence encoded in the HCV gene (see also Figure 22). It can be seen that other areas are relatively preserved. Even in the hyperchange domain, the 385 th threonine, the 386 th positive amino acid (arginine, histidine), the 389 th glycine, the 390 th glycine, the 402 leucine, the 403 th phenylalanine, the 404 th threonine And serine, 406th glycine, and 409th glutamine, have a high retention rate of 80% or more, with 387th threonine, 388th threonine, 392th alanine, 393th alanine, 394th positive electric amino acid. (Arginine, histidine), 395th threonine and serine, 396th threonine, 398th glycine, 399th leucine, 400th threonine, 401th serine, 407th proline, and 410th lysine and Arginine exhibits a retention rate of at least 50%.

A polypeptide comprising the hypervariable region can be expressed by inserting DNA having a nucleotide sequence encoding the same into a suitable prokaryotic or eukaryotic expression vector well known in the art (Sambrook et al., Molecular Cloning, A Laboratory Manual, 2nd ed., Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory Press, USA (1989).

The expression is preferably E. coli, eg, E. coli BL21 (DE3), E. coli JM109 (DE3), E. coli NM522, or yeast, e.g. Saccharomyces cerevisiae, Saccharomyces cerevisiae), Saccharomyces diastaticus and the like.

Suitable vectors that can be used for expression in E. coli and yeast are described in Sambrook et al. (Homologous) and in Fiers et al. (“Proced. 8th Int. Biotechnology Symposium”, Soc. Frac. De Microbiol., Paris , Durand et al., Eds., Pp. 680-697 (1988).

Transformation of host cells by the vector described above can be carried out by any of the conventional methods (Sambrook et al., Molecular Cloning, A Laboratory Manual (1989)). When transforming Escherichia coli, competent cells capable of absorbing DNA are prepared and then treated according to known CaCl ₂ methods. Transformation can also be carried out after forming the protoplasts of the host cells or by other methods as known in the art and described in Sambrook et al. (Homologous).

In general, the polypeptide can be prepared by culturing a host microorganism containing the desired expression vector under conditions enabling the expression of the DNA.

The polypeptide expressed in the transformed host cell can be precipitated from the cell culture or after cell disruption and / or precipitation, dialysis, ultrafiltration, gel filtration or ion-exchange chromatography, gel electrophoresis, for example with ammonium sulfate. Can be recovered by extraction by any suitable method known in the protein chemistry system, such as isoelectric point concentration, affinity chromatography, such as immunoaffinity chromatography, HPLC, and the like.

In addition, such polypeptides are useful antigens for HCV, can be used as diagnostic reagents or vaccines, or can be used to prepare monoclonal or polyclonal antibodies against these antigens, which can be used in the serum of hepatitis C patients. Use in detecting HCV antigens can greatly contribute to the diagnosis of HCV.

The M13 phage group containing the cDNA fragments (M13mp18-LBC-HV) was deposited with the ATCC on accession number ATCC75448 as of April 21, 1993.

Hereinafter, the present invention will be described in more detail based on 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) -4) unless otherwise specified.

Reference Example 1 Cleavage of DNA Using Restriction Enzymes

A restriction enzyme and a reaction buffer solution 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

10x NEB buffer 4: 200 mM tris-acetate, 100 mM magnesium

Acetate, 500mM potassium acetate, 10mM 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 saturation with 10 mM Tris-HCl (pH 8.0), 1 mM EDTA solution.

The sample and phenol were mixed at a ratio of 1: 1 (v / v) and then shaken vigorously, followed by centrifugation (15,000 × G, 5 minutes) to transfer the supernatant to a new tube. The same procedure was repeated three times, followed by extracting the supernatant with an equal volume of chloroform solution (chloroform to isobutanol ratio 24: 1 (v / v)), adding 0.1 volume of 3M sodium acetate and 2.5 volume of 100% ethanol. It was. After standing at −70 ° C. for 30 minutes or at −20 ° C. for at least about 12 hours, the resultant was centrifuged (15,000 × G, 20 minutes, 4 ° C.) to precipitate 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 dithiothreitol, 10 mM ATP) , 0.5 mg / ml BSA) was used to perform the ligation reaction. Usually, the reaction was performed at 16 ° C. for at least 2 hours using 10 unit ligation enzyme in a volume of 20 μl. After completion of the reaction, the ligation enzyme was inactivated by heating at 65 ° C. for 15 minutes.

Reference Example 4 E. coli Transformation

E. coli host cell JM101 (ATCC 33876) was inoculated into 100 ml of liquid LB medium (1% bactotrypton, 0.5% bacterium yeast extract, 0.5% sodium chloride) and the absorbance (OD) value at 650 nm was 0.25-0.5. Incubated at 37 ° C until reached. Cells were then separated by centrifugation (2,500 × G, 10 minutes), and then 50 ml of 0.1 M MgCl ₂ was added and washed, followed by centrifugation (2,500 × G, 10 minutes) and 50 ml of 0.1 M CaCl ₂ , 0.05M MgCl ₂ solution was added and allowed to stand on ice for 30 minutes. This was again centrifuged (2,500 × G, 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 coupling reaction solution was added to 0.2 ml of the solution 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 ampicillin and then incubated at 37 ° C. overnight. M13 vector DNA was subjected to heat treatment after 100 μl of E. coli JM101 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 2-fold soft solid YT medium (1% NaCl, 1% yeast extract, 1.6% bacto tryptone, 0.7% bacto agar) preheated to 48 ° C. Plated on YT medium (1% NaCl, 1% yeast extract, 1.6% bacto tryptone, 1.5% bactoagar).

Example 1. Preparation of cDNA

[1-1) Isolation of HCV RNA]

Serum of 21 Korean chronic hepatitis C patients with HCV identified by PCR (Sample A, Sample A1, Sample B, Sample 23, Sample 6, Sample 27B, Sample H1, Sample H4, Sample H5, Sample H6, Sample H12) , 300 μl of RNAzol B (Cinna / Biotecx; PO Box 1421, 100 μl of sample H19, sample H28, sample H34, sample HC6, sample L14, sample F1, sample F2, sample F3, sample I3, and sample I20). Friendswood, Texas USA) was added to lyse the cells. After the cells were lysed, 150 μl of chloroform was added, shaken vigorously for 15 seconds, then left on ice for 5 minutes, and centrifuged (12,000 × G, 4 ° C., 15 minutes). When the sample was divided into two layers, the supernatant was collected, the same volume of isopropanol was added, the mixture was allowed to stand at 4 ° C for 15 minutes, and centrifuged again (12,000xG, 4 ° C, 15 minutes) to precipitate RNA. The precipitated RNA was dissolved in 10 μl of diethylpyrocarbonate (DEPC) -treated distilled water containing 20 units of ribohexane degrading enzyme inhibitor (Promega) and immediately used to synthesize cDNA. Sample A, Sample A1, Sample B, Sample 23, Sample 6, Sample 27B, Sample H1, Sample H4, Sample H5, Sample H6, Sample H12, Sample H19, Sample H28, Sample H34, Sample HC6, Sample L14, and Sample F1 , HCV RNA extracted from Sample F2, Sample F3, Sample I3, and Sample I20 were respectively prepared as RNA-A, RNA-A1, RNA-B, RNA-23, RNA-6, RNA-27B, RNA-H1, RNA- H4, RNA-H5, RNA-H6, RNA-H12, RNA-H19, RNA-H28, RNA-H34, RNA-HC6, RNA-L14, RNA-F1, RNA-F2, RNA-F3, RNA-I3 and It was named RNA-I20.

[1-2) Preparation of cDNA]

3-4 μl of RNAs obtained in Example 1-1 were used as a template for MMLV reverse transcriptase, and cDNA Synthesis Kit (cDNA Synthesis Kit, Cat.No. 8267SA) of BRL (PO Box 6009, Gaithersburg, Md 20877 USA) CDNA was prepared. At this time, as a primer of the reverse transcriptase, random primers (5′-NNNNNN-3 ′, N means base G, A, T, and C are mixed in the same ratio) were used. To synthesize the first cDNA disconnection, 0.5 µl of 0.1M CH ₃ HgOH was added to 3-4 µl of the RNA solution obtained in Example 1-1), and left at room temperature for 10 minutes to release the secondary structure of RNA. 1 μl of 1M betamercaptoethanol was added and allowed to react for 5 minutes. 5 μl reverse transcriptase reaction solution (250 mM Tris-HCl, pH 8.3, 375 mM KCl, 15 mM MgCl ₂ , 5 mM dithiothreitol, 1.25 μl of 10 mM dNTP (dATP, dGTP, dTTP, dCTP), 1 μl of random DEPC (diethylpyrocarbonate) -treated distilled water was added to the primer (1.0 g / μl) and 1.0 μl of the RNase inhibitor (1 U / μl, Promega, USA) so that the total volume was 25 μl. The RNA template and the primer were attached to each other, and 1.25 μl of superscript H ^- reverse transcriptase (18 U / μl; BRL) was added and reacted at 37 ° C. for 1 hour to synthesize cDNA helix. CDNA-A, cDNA-A1, cDNA-B, cDNA-23, cDNA-6, cDNA-27B, cDNA-H1, cDNA-H4, cDNA-H5, cDNA-H6, cDNA-H12, cDNA-H19, cDNA, respectively -28, cDNA-H34, cDNA-HC6, cDNA-L14, cDNA-F1, cDNA-F2, cDNA-F3, cDNA-I3 and cDNA-I20.

[Example 2. cDNA Amplification of HCV by Polymerase Chain Reaction]

[2-1) Design of Primer]

In order to amplify HCV cDNA in the HV region, nucleotide sequence information of a previously published Japanese hepatitis C virus (Kato et al., Proc. Natl. Acad. Sci. USA 87, 9524-9528 (1990); Takamizawa et al., J. Virol. 65, 1105-1113 (1991)), nucleotide sequence information of US hepatitis C virus (Choo et al., Science 244, 359-363 (1989)) and Korean hepatitis C virus With reference to the nucleotide sequence information (prior Korean patent application No. 92-10039 of the applicant), the nucleotide sequence was relatively selected and the following primer pairs for PCR were devised. The nucleotide sequence and position of the prepared primers are based on the nucleotide sequence of KHCV-LBC1 described in patent application No. 92-10039.

HVBAM 5′-GGTCACCGGATCCATGGCCTGGGATATGATGATGAA-3 ′

This primer has a restriction enzyme BamHI recognition site at the 5 'end and corresponds to the upper strand nucleotide sequence containing the 1294th to 1316th nucleotides of KHCV-LBC1.

HVSAL 5′-CCGTCGACATTCATCCATGTACAACCGAACCA-3 ′

This primer has a restriction enzyme SalI recognition site at the 5 'end and corresponds to the lower strand nucleotide sequence containing the 2010 to 1987 nucleotides of KHCV-LBC1.

The primers designed as above were synthesized by a DNA synthesizer (Applied Biosystems, Inc., model 380 B, USA) using an automated solid phase phosphoamidite chemistry, and then modified polyacrylamide gel (2M). Urea, 12% acrylamide: separated by electrophoresis using bis (29: 1, w / w), 50 mM Tris, 50 mM boric acid, 1 mM EDTA-Na. It was attached to C18 column SEP-PAK (Waters Inc., U.S.A.), eluted with 50%: 50% acetonitrile: water mixture, and purified purely. The concentration was determined by measuring absorbance at 260 nm.

[2-2) Polymerase Chain Reaction (PCR)]

PCR reaction was carried out as follows. 5 μl of HCV cDNA obtained in 1-2) as a template, 10 μl of 10-fold concentration Taq polymerase buffer solution (10 mM Tris-Cl pH8.3, 500 mM KCl, 15 mM MgCl ₂ , 0.1% (w / v) gelatin), Distilled water was added to 10 μl of dNTP mixture (1.25 mM of dGTP, dATP, dTTP, dCTP, 0.2 μg each of starting HVBAM and starting HVSAL) and 1 μl (2.5 units) of AmpliTaq DNA polymerase (Perkin Elmer Cetus, USA). The total volume was added to 100 µl. To this was added 50 μl of mineral oil to prevent evaporation of the solution. A temperature cycler (Thermer Cycler; Perkin-Elmer Cetus, USA) was used and the cycle program was 95 ° C., 2 minutes; 55 ° C., 2 minutes; The cycle of 72 ° C., 3 minutes was repeated 40 to 45 times. In order to remove the polymerase after the reaction, the same volume of phenol / chloroform was added to the reaction mixture, the mixture was mixed well, and centrifuged. The supernatant was transferred to a new tube, mixed with 1/10 volume of 3M sodium acetate, 2.5 times volume of 100% ethanol, and centrifuged to obtain amplified double helix hexane, which was added to 20 μl of TE buffer (10 mM Tris-HCl). , pH 7.5, 1mM EDTA) was used in the next experiment.

The amplified cDNAs were divided into fragments HV-A, HV-A1, HV-B, HV-23, HV-6, HV-27B, HV-H1, HV-H4, HV-H5, HV-H6, HV-H12, HV-H19, HV-H28, HV-H34, HV-HC6, HV-L14, HV-F1, HV-F2, HV-F3, HV-I3 and HV-I20.

The amplified cDNAs were separated by polyacrylamide gel electrophoresis, respectively, and digested with restriction enzymes BamHI and SalI in the same manner as in Reference Example 1.

After cleavage of the cleaved cDNAs to M13mp18, the nucleotide sequence was determined according to Sanger's dideoxy method and the amino acid sequence was inferred therefrom (see FIGS. 1 to 21). Comparison of the inferred amino acid sequences showed significant changes from the 384th amino acid to the 410th amino acid (see FIG. 22) and the other regions were relatively conserved. The comparison of amino acid sequences in FIG. 22 is from 316 th amino acid to 420 th amino acid, and their consensus amino acid sequence (which means the most frequently occurring amino acid sequence) is YYSMVGNWAKVLIVMLLFAGVDGTTHTTGGTAARTTSGLTSLFSPGPAQKIQLINTNGSW, in particular the hyperchange region (384 th amino acid). Consensus amino acid sequence from 410th amino acid) is TTHTTGGTAARTTSGLTSLFSPGPAQK. "-" Represents the same amino acid as the common amino acid and "/" represents the deleted amino acid.

The degree of expression of specific amino acids or amino acids in the hypervariable region of 21 different clones and 21 clones of KHCV-LBC1 found in the present invention from FIG. 22 is expressed as a percentage.

385th: threonine (90%)

386th: positive positive amino acids (arginine, histidine; 81%)

387th: Threonine (57%), Valine (29%)

388th: threonine (57%), valine (29%)

389th: Glycine (90%)

390th: Glycine (100%)

391th: Threonine and Serine (48%), Alanine (24%)

392th: Alanine (52%)

393th: Alanine (62%), Glycine (19%)

394th: positively charged amino acids (arginine, histidine; 67%)

395th: threonine and serine (57%), alanine (24%)

396th: threonine (67%), alanine (19%)

397th: serine (33%), positively charged amino acids (arginine, lysine and histidine; 38%)

398th: Glycine (52%), Threonine and Serine (29%)

399th: Leucine (62%), Phenylalanine (29%)

400th: threonine (71%), alanine (29%)

401st: Serine (76%)

402th: Leucine (86%)

403th: Phenylalanine (95%)

404th: threonine and serine (86%)

406th: Glycine (95%)

407th: Proline (62%), Alanine (24%)

408th: Alanine (38%), Serine (38%), Glutamine (19%)

409th: Glutamine (86%)

410th: Lysine and arginine (62%), asparagine (33%)

Claims (28)

One or more amino acids from amino acids 384 to 410 (based on the total amino acid sequence coded for the hepatitis C virus gene), the hypervariable region of the E2 / NS1 protein amino acid sequence of the Korean hepatitis C virus (KHCV) KHCV polypeptide comprising a substituted sequence: threonine at 385 amino acid, arginine or histidine at 386 amino acid, threonine or valine at 387 amino acid, threonine or valine at 387 amino acid, glycine at 389 amino acid , 390th amino acid is glycine, 391th amino acid is threonine, serine or alanine, 392th amino acid is alanine, 393th amino acid is alanine or glycine, 394th amino acid is arginine or histidine, 395th amino acid is threonine , Serine or alanine, the 396th amino acid Threonine or alanine, 397 amino acid serine, arginine, lysine or histidine, 398 amino acid glycine, threonine or serine, 399 amino acid leucine or phenylalanine, 400th amino acid threonine or alanine, 401 th amino acid Amino acid serine, 402th amino acid leucine, 403th amino acid phenylalanine, 404th amino acid threonine or serine, 406th amino acid glycine, 407th amino acid proline or alanine, 408th amino acid alanine , Serine or glutamine, 409th amino acid is substituted with glutamine, 410th amino acid is substituted with lysine, arginine or asparagine. The polypeptide of claim 1 having the following amino acid sequence. A K V L I V M L L F A G V D G T T Y T T G G T T G R T T G G L T S L F T S G A S Q K V Q L V N T N G S W H I N R T A L N C N D S L Q T G F L A A L F Y T H R F N S S G C Q R The polypeptide of claim 1 having the following amino acid sequence. N W S P T A A L V M S Q L L R I P Q T V L D V V A G A H W G V L A G L A Y Y S M A G N W A K V L I V L L L F A G V D G G E A W R T R T I G G K Q P Q G V S T I T S L F S S G P A Q K I Q L V N T N G S W H I N R T A L N C N D S F Q T G F L A A L F Y V R N F N A S G C P E R M A S C R P I D T F N Q G W G P I T Y T R P D S P D Q R P Y C W H Y P The polypeptide of claim 1 having the following amino acid sequence. M V G N W A K V L I V M L L F A G V D G T T V T M G G T V A R T T T G F T F L F R P G A S Q K I Q L I N T N G S W H I N R T A L N C N D F L N T G F L A A L F Y T H R F N A S G C P E R M A S C Q S I D R F I Q G W G P I T Y A E N G S S D Q R P Y C W H Y P P The polypeptide of claim 1 having the following amino acid sequence. A K V L I V M L L F A G V D G E T H V T G G S V A G Y T S G L T S L F T L G P A Q K I Q L V N T N G S W H I N R T A L N C N D S L H T G F L A G L F Y H H R F N A S G C P A R M A S C R P I D K F D Q G W G P I T Y A K Q G S Q D Q R P Y C W H The polypeptide of claim 1 having the following amino acid sequence. G S S S C D T T R A V V G D Q F I I I S Q A M R I P Q A I V D M V A G A H W G V L A G L A Y Y S M V G N W A K V L I V M L L F A G V D G T A R T T G G A A V P S T S G V A S L F I S G P A Q R I Q L I N T N G S W H I N R T A L N C N D S L Q A G F L A A L F Y A H K F N A S G C P E R M A S C R S I D A F D Q G W G P I T Y T The polypeptide of claim 1 having the following amino acid sequence. A K V L I V M L L F A G V D G R T R T V G G T A A R D T S G F A S L F T A G P A Q K I Q L V N T N G S W H I N R T A L N C N D S L Q T G F L A A L F Y T R N F N A S G C L E R M A S C R P I G A F D Q G W G P I T Y A E R R G S D Q R P Y C W H Y P The polypeptide of claim 1 having the following amino acid sequence. N W S P T T A L V V S Q L L R I P R A I M D V V A G A H W G V L A G L A Y Y S M V G N W A K V L I V M L L F A G V D G G T Y T T G G T Q A R A A H G F T S L L S L G A S Q K V Q L I N T N G S W H I N R T A L N C N E S L Q T G W L A A L F Y T H K F N A S G C P E R M A R S A A I D T F N Q G W G P V T Y T E P G S R D Q K P Y C R H Y A P R Q C G I A P A S E V C V Q S T The polypeptide of claim 1 having the following amino acid sequence. N W S P T T T I I L A Y V M R V P E V I I D I I S G A H W G V M F G L A Y F S M Q G A W A K V V V I L L L A A G V D A R T H T S G G S V A R A T Y G L T S W F S P G A R K C Q L I N T N G S W H I N R T A L N C N D S L H T G F I A S F The polypeptide of claim 1 having the following amino acid sequence. N W S P T T A L V V S Q L L R I P Q A I M D M M A G A H W G V L A G L A Y Y S M V G N W A K V L I V M L L F A G V D G A T Y T T G G A Q A A H G L T S L L S R G A S Q R V Q L I N T N G S W G I N R T A L N C N E S L Q T G W L A A L F Y T H K F N A S G C P E R M A R C R P I D T F D Q G W R P I T Y T E P G D L D Q R S Y C W H Y A P R Q C S I V P A S Q V The polypeptide of claim 1 having the following amino acid sequence. N W S P T S T M I L A Y V M R V P E V I I D I I S G A H W G V M F G L A Y F S M Q G A W A K V V V I L L L A A G V D A S T H T T G G S A T Y T T S S L A K M F S F G S N Q N I Q L I N T N G S W H The polypeptide of claim 1 having the following amino acid sequence. N W S P T T A L V V S Q L L R I P Q A V L D M V A G A H W G V L A G L A Y Y S M V G N W A K V L I V M L L F A S V D G S T R V T G G R H A H T T K S L A S L F S S G P S Q K I Q L V N T N G S W H I N R T A L N C N D S L Q T G F The polypeptide of claim 1 having the following amino acid sequence. N W S P T A T M I L A Y A M R V P E V I I D I I S G A H W G V M F G L A Y F S M Q G A W A K V V V I L L L T A G V D A N T H M V G G S A A R S T R G F T S L F S S G P Q R N I Q L I N T N G S W G I N R T A L N C N E S The polypeptide of claim 1 having the following amino acid sequence. N W S P T A T M I L A Y A M R V P E V I I D I I S G A H W G V M F G L A Y F F M Q G A W A K V V V I L L L T A G V D A D T H A V G G A A A R S A Y R L T S F F S L G P Q Q N I Q L I N T N G S W H I N R T A L N C N D S L N T The polypeptide of claim 1 having the following amino acid sequence. N W S P T A T M I L A Y S M R I P E V I I D I I S G A H W G V M F G L A Y F S M Q G A W A K V A V I L L L A A G V D A H T H V A G G T A A F N A H G L A S L F T R G A Q Q K T Q L I N T N G S W H V N R T A L N C N D S L R T G F I A The polypeptide of claim 1 having the following amino acid sequence. N W S P T A A L V V S Q L L R I P Q A V M D M L A G A H W G V L A G L A Y Y S M V G N W A K V L I V M L L F A G V D G E T R V T G G R A G Q T T R S L T S L F S T G P Q Q K I Q L I N T N G S W H I N S T A L D C N D S L Q T G F L A A R F Y The polypeptide of claim 1 having the following amino acid sequence. N W S P T T A L V V S Q L L R I P Q A V V D M V T G S H W G I L A G L A Y Y S M V G N W A K V L I V M L L F A G V D G T A Y E D T T P S H Q G G R P A A L Q V H V L F D L G P S Q K V Q L I N T N G S R H I N R T A L N C N D S L N T G F I A S L F S T Y R F N A S G C P A R L A S C D P I E T V A Q G W G P I T Y G E P A D S D Q R P Y C W H Y A P R Q C G I V P A S E V C G P The polypeptide of claim 1 having the following amino acid sequence. N W S P T A A L M V S Q L L R I P Q A I V D M V A G A H W G V L A G L A Y Y S M V G N W A K V L I V M L L F A G V D G T T H T T G G S V A R S T S K L T C L F S A G SA Q N I Q L M The polypeptide of claim 1 having the following amino acid sequence. L V A G A H W R V L A G L A Y Y S M M G N W A K V L I V M L L F A G V D G E T H T T G G A A A H S T R T L T S L F T S G P A Q R I Q L I N T N G S L H I N R T A L N C N D T L Q T G F F A A L F Y The polypeptide of claim 1 having the following amino acid sequence. N W S P T A T M I L A Y A M R V P E V I I D I I S G A H W G V T F G L A Y F S M Q G A W A K V V V I L V L A A G V D A G T H T V G G Q A A G S V Y R F T G L F T R G A K T N I Q L V S S The polypeptide of claim 1 having the following amino acid sequence. N W S P T A T M I L A Y V M R A P E V I I D I V S G A H W G V M F G L A Y F S M Q G A W A K V V V I L L L T A G V D A Q P R I T A Q A A H A T S G L A N L F T P G S Q Q N I Q L I S T N G S W The polypeptide of claim 1 having the following amino acid sequence. N W S P T A A L V V S Q L L R I P Q A V M D V I A G A H W G I L A G L A Y Y S M V G N W A K V L I V M L L F A G V D G H T R V T R G V Q G R T T A G L T S L F S P G P A Q N I Q L DNA comprising a base sequence for coding the polypeptide of claim 1. The DNA of claim 23 comprising a nucleotide sequence encoding a polypeptide of any one of claims 2-22. The DNA of claim 24 comprising any of the nucleotide sequences of FIGS. 1 to 21. An expression vector comprising the DNA of claim 23. A microorganism transformed with the vector of claim 26. A method of producing the polypeptide of any one of claims 1 and 2 to 22 by culturing the microorganism of claim 27.