KR100269803B1 - Expression of antigen determining site-fusion protein of hepatitis c virus envelope protein - Google Patents

Abstract

PURPOSE: A fusion protein of epitope in envelope protein of hepatitis C virus to diagnose hepatitis C more correctly and cheaply is provided, which contributes to the development of reagents and vaccines against HCV. CONSTITUTION: The fusion protein of epitope in envelope protein of hepatitis C virus is manufactured by the next steps of: i) amplifying epitope genes in envelope protein of hepatitis C virus through PCR and purifying amplified DNA segments to obtain DNA segment, E1D2; ii) making an expression vector for E. coli; iii) transforming E. coli W3110 (ATCC 37339) to manufacture recombinant E. coli having ptrpH-UB-E1E2 inserted with DNA segment E1E2; iv) cultivating the strain in broth and centrifuging it for 25 minutes at 11,000rpm to obtain cell sediments; and v) measuring the expression of protein UBE1E2 and reacting the protein with serum of hepatitis C patients.

Description

Expression of Antigen Decision-site Fusion Proteins in Envelope Proteins of Hepatitis C Virus

Figure 1 shows the nucleotide sequence and amino acid sequence of the E1G, E2A, E2E gene encoding the antigenic determining region of the envelope protein of hepatitis C virus,

2 shows an expression vector comprising the linking strategy of the E1G, E2A and E2E genes and the linker gene UBE1E2 with ubiquitin,

3 (a) shows the results of confirming the expression of EBE1E2 protein, which is a recombinant protein of the ubiquitin and the antigenic determinant of the envelope protein, by SDS polyacrylamide gel electrophoresis.

Figure 3 (b) shows the results of Western blotting the UBE1E2 recombinant protein to the serum of hepatitis C patients.

The present invention provides an antigen-determining site fusion protein of an envelope protein of hepatitis C virus (hereinafter referred to as "HCV"), an expression vector for producing the same, and an antigen-determining site of HCV envelope protein using a microorganism transformed with the vector. A method for producing a protein.

More specifically, HCV base sequences 1201 to 1509 309 base pairs corresponding to the carboxyl terminus of the envelope 1 protein, the antigenic determining region of the envelope protein of HCV, and HCV bases corresponding to the amino terminus of the envelope 2 protein. The antigenic determinant of the envelope protein of HCV upon expression thereof, including the binding gene of the genes of the HCV base sequences 2281-2529 and the 249 base pair genes corresponding to the carboxyl termini of the envelope 2 protein of SEQ ID NOs: 1510-1749 Method for producing large amount of fusion protein antigen-determining site of HCV envelope protein using expression vector to produce fusion protein, microorganism transformed with the expression vector and antigen-determining site of HCV envelope protein fusion protein It is about.

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). Unlike the above hepatitis, non-AB hepatitis B (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 approximately 1 million people worldwide are infected with this non-A hepatitis B virus (Alter, HJ, in AJ Zuckerman (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 non-B-type virus (hereinafter referred to as HCV) can be isolated and recovered by infecting chimpanzees with NANBH patients (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 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 flaviviruses and pestiviruses, and due to their flexibility, the polyprotein of hepatitis C virus is expressed from the amino terminus to the nucleus. core-envelope 1-envelope 2 / unstructure 1 (E2 / NS1) -unstructure 2 (NS2) -unstructure 3 (NS3) -unstructure 4 (NS4) -unstructure 5 (NS5) (Choo, QL, et al., Proc. Natl. Aca. Sci. USA 88, 2451-2455 (1991); Takamizawa, A., et at, 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 shown to be glycoproteins with molecular weights of 33,000 and 72,000 daltons, respectively (Houghton, M., et al., Hepatology 14, 381-388 (1791); Hijkata, M., et al., Proc. Natl. Acad. Sci USA 88, 5547-5551 (1991)), presumed to be vaccines of flaviviruses Significant structural similarity to the gp53 / 55 protein, which is believed to be a vaccine of structure 1 protein and pestivirus, has been the primary goal for vaccine development and treatment of 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 the viral ribonucleic acid polymerase so far known, and is estimated to be the RNA polymerase of the hepatitis C virus (Mita, E., et at., 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 have antibodies to this antigen (Kuo, G., et al., Soience 244, 362-384 ( 1989). However, in many patients, antibodies against C100-3 are produced four to six months after hepatitis C virus infection, making it impossible to accurately diagnose 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. Hepatitis patients have been reported to have a positive rate of positive reactions (Mc Fararlane, IG, et. Al., Lancet 335, 754-757 (1990)).

Recently, the diagnostic method using a nuclear protein expressed from the core structural gene located at the 5 ′ end of the 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)), and the existing C100-3 antigen. The second-generation diagnostic method, in which C22 derived from nuclear structural genes and C33C derived from non-structural 3 genes is added, has been reported to further increase the sensitivity and specificity for HCV antibodies (Van der poel, CL, et. al., Lancet 337, 377-319 (1991).

The present inventors have isolated HCV particles from the sera of Korean hepatitis C patients and identified their genetic structure to identify unique Korean HCVs that differ from those of US and Japanese HCVs, and KHCV from nuclear genes. KHCV UB897 protein from UB Core14, non-structural 3 gene and KHCV 403 protein from non-structural 5 gene were derived from recombinant yeast and Escherichia coli (prior Korean patent applications 91-10942, 91-10943, 91-25505) And 92-10039), a method of identifying and immunomodifying these proteins and then mass separation and purification with high purity has been developed (prior Korean Patent Applications 91-13594, 91-13595, 91- 13597, 91-25506 and 92-10039). Using these HCV specific antigens, we have also developed and reported an improved diagnostic method for assaying antibodies against hepatitis C virus from the serum of patients according to known ELISA immunoassay (EIA) principles. See Korean Patent Application No. 91-13601.

In addition, it was found that the protein product of the amino terminal 1,200 base pairs (non-structural 5-1.2 protein) of the non-structural 5 gene including the KHCV 403 protein exhibited a high specific immune response with the antibody of HCV and ubiquitin It was expressed in recombinant E. coli as a fusion protein (UB NS5-1.2) with (UB) (see preceding Korean Patent Application No. 93-4165), and a partial sequence of the non-structural 4 gene in the entire gene of Korean HCV was Korean hepatitis C. It was confirmed that the reaction with the patient's serum and immunological specificity, and expressed it as a fusion protein (UB NS4E) with ubiquitin in recombinant E. coli by genetic engineering method (see the preceding Korean Patent Application No. 93-4164).

In these antigenic proteins, the antigenic epitope of HCV, which specifically binds antibodies in the serum of hepatitis C patients, is 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 specificity 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 reaction with the serum of the hepatitis C patient 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 total gene were expressed in E. coli. By expressing and measuring the response of the hepatitis C patient to the serum, it was found that the antigenic determinant is located within the 222 base pairs encoding the amino terminus (Nasoft, MS et al., Proc. Natl. Acad Sci. USA). 88, 5462-5466 (1991).

The present inventors measured the reaction of hepatitis C patients with E. coli expressing 8 different gene fragments of KHCV897 protein derived from the nonstructural 3 gene of HCV. (See Korean Patent Application No. Applicant filed 15 April 1993 by Applicant), and in the case of coat protein, the antigenic determinant is from 309 base pairs corresponding to HCV sequences 1201 to 1509. Carboxyl terminus of the expressed envelope 1 protein; The amino terminus of the envelope 2 protein expressed from 240 base pairs corresponding to HCV sequences 1510 to 1749; And the carboxyl terminus of the envelope 2 protein expressed from the 249 base pairs corresponding to HCV base sequences 2281 to 2529 (see Korean Patent Application No. 93-5663 filed by the applicant).

On the other hand, specific antigens of purely isolated HCV can be used to prepare monoclonal or polyclonal antibodies against these antigens and by using these antibodies to detect HCV antigen in the serum of hepatitis C patients. Antibodies that can significantly contribute to more sensitive early diagnosis, especially those that have specificity for neutralizing epitopes, are also useful for passive immunotherapy. Monoclonal antibodies can also be used to increase anti-idiotype antibodies, which are known to be useful in the treatment and diagnosis of hepatitis C, as well as in identifying immunogenic regions of HCV antigens.

Ubiquitin, meanwhile, was a protein with a molecular weight of 5,500 Daltons, which was discovered in 1975, and is found in eukaryotic cells as a whole and without its genes (Goldstein, et al., Proc. Natl. Acad. Sci 72, 11 (1975). )). So far, the ubiquitin found in animal cells has been 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 gene products expressed as ubiquitin fusion proteins in cells, without being degraded. Such a fusion protein system is particularly useful because the ubiquitin fused to protect the protein to be easily degraded by protease in the cell, and the expression can be confirmed 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 at., J. Biological Chemistry 266, 12021-). 12028 (1991).

Based on this fact, the present inventors have diagnosed Korean HCV with higher sensitivity and specificity than those using proteins encoded by US-type and Japanese-type HCV. In an effort to develop reagents and vaccines, E1G, E2A, and E2E genes encoding antigenic determinants of the envelope protein of Korean HCV were combined and successfully expressed as a fusion protein in Escherichia coli by genetic engineering method. Was completed.

It is an object of the present invention to provide an antigenic determinant fusion protein of the envelope protein of HCV which may contribute to the development of diagnostic reagents and vaccines with higher specificity for Korean HCV.

It is another object of the present invention to provide an expression vector which comprises an antigen-binding gene of a gene encoding an antigenic determinant of an envelope protein of HCV and, upon its expression, produces an antigenic determinant fusion protein of an envelope protein of HCV.

It is still another object of the present invention to provide a microorganism transformed with the expression vector and a method for producing a large amount of the epitope fusion protein of the envelope protein of HCV using the microorganism.

Hereinafter, the present invention will be described in detail.

In the development of a hepatitis C diagnostic reagent, it is preferable to use an antigen protein having high specificity for the hepatitis C antibody, and its antigen determination site is more preferable since it enables the development of an efficient and economical diagnostic reagent and vaccine. In particular, an antigen-binding site fusion protein in which several epitopes are fused is most preferable in terms of economic efficiency, efficiency and accuracy. As such epitope fusion protein, E1E2, which is an epitope fusion protein of the present invention containing three epitopes of the envelope protein of hepatitis C virus, is preferable. In addition, the fusion protein can be produced as a recombinant fusion protein by further fusion of a specific protein for stabilization, confirmation of expression and simple purification of the protein, and ubiquitin is preferred as such a specific protein.

First, the sequence of the E1G, E2A, and E2E genes encoding the antigenic determinant protein of the envelope gene was obtained from sequencing information on cDNA fragments of the Korean hepatitis C virus (see Korean Patent Application No. 92-10039, previously filed by the applicant). The primers for the polymerase chain reaction (PCR) corresponding to the 5 'end and the 3' end were synthesized. These primers are designed to connect the E1G, E2A, and E2E genes to each other, and the restriction enzyme recognition sites are present at the 5 'end to allow conjugation with the 3' end of a specific protein gene. The corresponding primers have termination codons and restriction enzymes. By inserting the recognition site of the protein is synthesized from the start codon of the specific protein to complete the protein synthesis in the artificial coinsertion termination codon and facilitates cloning into the expression vector. Using these primers KHCV-LBCl (deposited accession number ATCC 75008 dated May 14, 1991) of cDNA of the hepatitis C virus (see Korean Patent Application No. 92-10039, filed by the applicant) or shell 1 and Gene fragment obtained by amplifying E1G, E2A, and E2E binding genes (E1E2) by polymerase chain reaction using envelope 2 gene (see Korean Patent Application No. 92-10039) as a template, and digesting this amplified gene with restriction enzyme Was substituted with the gene following the specific protein of the plasmid containing the gene of the specific protein to prepare an expression vector. The envelope antigen-determining site fusion protein expressed from the above expression vector was electrophoresed on 15% polyacrylamide gel, and then Western-blotted with immunoglobulins isolated from the serum of Korean hepatitis C patients, and the UBE1E2 protein was Korean-type C. It was confirmed that the antibody specifically reacts with an antibody of hepatitis virus.

When the antigen-determining site fusion protein of the present invention is used for the production of HCV diagnostic kits and vaccines, it is more economical than using multiple types of HCV antigens, including several antigen-determining sites but using only a single protein. Phosphorus diagnostic kit or vaccine can be prepared, and since only the fusion regions of HCV antigens are fused, it is possible to prepare a more sensitive and accurate diagnosis and efficient vaccine compared to the case of simply fusion of existing HCV antigens.

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 in 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 saturation 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, mixed, and centrifuged (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 3H sodium acetate and 2.5 volume of 100% ethanol. do. This was allowed to stand at -70 ° C for 30 minutes or at -20 ° C for at least about 2 hours, followed by centrifugation (15,000 x G, 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 (0.5 M Tris-HCl, pH 7.8, 0.1 M MgCl ₂ , 0.2 M dithiothreitol, 10 mM ATP, Ligation reaction was carried out using 0.5 mg / ml BSA). Usually, 10 units of ligase is used in a volume of 20 μl, and 100 units of ligase are 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]

E. coli host cells (E. coli HB101, W3110 or JM105) were inoculated into 100 ml of liquid LB medium (1% bactotrypton, 0.5% bacterium yeast extract, 0.5% sodium chloride) and the optical density (OD) value at 650 nm was 0.25 Incubate at 37 ° C. until reaching 0.5. Cells are then separated by centrifugation (2,500 x G, 10 minutes) and then washed by adding 50 ml of 0.1 M MgCl ₂ . This was again centrifuged (2,500 × G, 10 minutes) and 50 ml 0.1M CaCl ₂ , 0.05M MgCl ₂ , a solution was added thereto, 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 heat treated and then 100 μl of 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 double soft solid YT medium (1% NaCl, 1% yeast extract, 1 6% bacto tryptone, 8.7% bacto agar) preheated to 48 ° C. Plated on YT medium (1% NaCl, 1% yeast extract, 1.6% bacto tryptone, 1.5% bacto agar).

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% acrylic). Amide, bis (29: 1 w / w) in 50 mM Tris, 50 mM boric acid, 1 mM EDTA-Na ₂ ) was separated by electrophoresis. Next, it was attached to a Cl8 column, SEP-PAK (Waters Inc., USA), and purified using pure acetonitrile: water (50:50) as an eluent. The concentration was determined by measuring absorbance at 260 mm.

Reference Example 6

[Po1ymerase Chain Reaction (PCR)]

Template DNA (10 ng to 100 ng), 10 μg of 10-fold Taq polymerase buffer (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 (dGTP , dATP, dTTP, dCTP each 10 mM), distilled water was added to 2 μg, 0.5 μl AmpliTaq DNA polymerase (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. The temperature cycler (Perkin Elmer Cetus, USA) was used, the cycle program was repeated 1 minute at 95 ℃, 1 minute at 55 ℃ 2 minutes at 72 ℃ and finally the reaction was further reacted at 72 ℃ 10 minutes.

Example 1

[Amplification of the Antigen Determination Site Binding Gene in the Envelope of Korean Hepatitis C Virus]

[Step 1]

The antigen-binding site-binding gene of the envelope, which is part of the cDNA (KHCV-LBCl, Korean Patent Application No. 92-10039, ATCC 75008, deposited May 14, 1991) of Korean hepatitis C virus cloned previously To clone into the E. coli expression vector having a trp promoter coupled to ubiquitin, the following primers were synthesized.

PE2ET2 5′-TGAGACTCCGCGGTGGTACTCGGGGAGAGCGTTGTGAC-3 ′

This primer has a restriction enzyme Sac II recognition site and contains the bases 2281 to 2298 of KHCV-LBCl, the cDNA of Korean hepatitis C virus. This primer is

PE2EGE1G 5′TTCCTTCTTCTGGCGGACGCGGTTTCCCAGCTGTTCACCTTC-3 ′

This primer contains the 2529th base from the 2509th base of KHCV-LBCl which is the 3 'end of E2E, and the 1201st-1221th base of the KHCV-LBCl which is the 5' end of E1G gene.

PE2AXHO 5′-AAAAAACTCGAGTTACCACCCCTGCGCGAATGTATC-3 ′

This primer has a terminating codon that terminates translation after the 1749th base of KHCV-LBCl and at the same time has a restriction enzyme Xhol recognition site.

[Step 2]

2μg of primer PE2EGE1G and 2μg of primer PE2AXHO were added to the reaction tube, and 50ng of KHCV-LBCl cDAN was added as a template.10μl of 10-fold polymerase buffer solution, 10μl of 2mM dNTP (2mM dGTP, 2mM dATP, 2mM dCTP), 2.5 units of Taq polymerase and distilled water were added to make the total volume 100 μl, and the polymerase chain was treated at 95 ° C. for 30 seconds, at 55 ° C. for 30 seconds, and at 72 ° C. for 1 minute as in Reference Example 6. The reaction was performed 25 times (Saiki, RK et. Al., Science 239, 487 (1988)).

[Step 3]

The PCR product obtained in step 2 was isolated from 5% polyacrylamide gel, and it was confirmed that about 550 base pairs of DHA were amplified, and this was separated and purified by polyacrylamide gel under the same conditions. This DNA fragment was named fragment GE1GE2A.

[Step 4]

2 μg of the primer PE2ET2 and 2 μg of the primer PE2AXHO were added to the reaction tube, and 50ng of the plasmid pYLBC-A / G-UBE2C (Korean Patent Application No. 92-10039, ATCC 74117) and 50ng of the fragment GE1GE2A obtained in step 2 were added as a template. 10 μl of 10-fold polymerase buffer solution, 10 μl of 2 mM dNTP (2 mM dGTP, 2 mM dATP, 2 mM dTTP, 2 mM dCTP), 2.5 units of Taq polymerase and distilled water were added to make the total volume 100 μl. As in 6, 25 polymerase chain reactions were performed for 30 seconds at 95 ° C, 30 seconds at 55 ° C, and 1 minute at 72 ° C.

[Step 5]

The PCR product obtained in step 4 was isolated from 5% polyacrylamide gel, and it was confirmed that about 800 base pairs of DNA was amplified and separated and purified by polyacrylamide gel under the same conditions. This DNA fragment was named Fragment E1E2.

Example 2

[Production of Escherichia Coli Expression Vector]

2 μg of plasmid ptrpH-UB-CORE14 (Korean Patent Application No. 91-13603, ATCC 68642, dated Jul. 1, 1991) filed by the Applicant with NEB buffer solution with restriction enzyme Sac II and restriction enzyme Sal I Under the conditions of 3, after complete cleavage as in Reference Example 1, the resultant was separated and purified from 0.7% agarose gel to separate and purify about 2.7 kb fragment. This fragment was named ptrpH-UB-T2 / L.

Meanwhile, 2 μg of the DNA fragment obtained in step 3 of Example 1 was completely cleaved with restriction enzyme Sac II and restriction enzyme Xho I under the conditions of NEB buffer 3 as in Reference Example 1, and then 100 ng, 50 ng of the DNA fragment was cut into the tube. Fragment ptrpH-UB-T2 / L, 2 μl of 10-fold conjugation reaction solution, 10 units of T4 DNA ligase were added, distilled water was added to a total volume of 20 μl and reacted at 16 ° C. for 12 hours. After the reaction, E. coil W3110 (ATCC 37339) was used to transform the recombinant E. coli containing ptrpH-UB-E1E2 containing fragment E1E2 (see FIG. 2).

Example 3

[Expression of E1E2 gene, which is the binding region of the antigenic determining region of the envelope]

Recombinant Escherichia coli cells containing the E1E2 gene, which is an antigen-determining site-binding gene of the envelope of hepatitis C virus, obtained in Example 2, were cultured in a liquid Luria medium containing 50 μg / ml of ampicillin (6% bactotrypton). After shaking for 12 hours with 0.5% yeast extract and 1% sodium chloride, 3 ml each of 300 ml of M9 medium (40 mM K ₂ HPO ₄ , 22 mM KH ₂ PO ₄ , 8.5 mM NaCl, 18.7 mM NH _{4) was added.} Cl, 1% 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 for about 4 hours at 37 ° C. When the absorbance was about 0.3 at a wavelength of 650 nm, 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 the 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

[Confirmation of UBE1E2 Fusion Protein Expression and Response with Hepatitis C Patient Serum]

The cell precipitate of Example 3 was electrophoresed on a 15% polyacrylamide gel in the presence of SDS according to the method of Laemmli (Laemmli, Nature 227, 680 (1970)) (Fig. 3 (a)) and isolated on the gel. Proteins were nitrocellulose filters (Bio-Rad Lab., Pore size 0.22 μm, CA, USA) according to Tobin's method (Towbin, et al., Proc. Natl. Acad. Sci. USA 76, 4750 (1979)). Delivered to. 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 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. Anti-human immunoglobulin G (Bio-Rad Lab, anti-human IgG-HRP, 0.01 mg / m1 labeled with horseradish peroxidase in PBS containing 0.5% gelatin and 0.05% Tween 20 in this filter) , CA, USA) was added at a dilution of 1: 500 and reacted at room temperature for 1 hour by shaking, washed 4 times with PBS containing 0.2% Tween 20 for 5 minutes, and then twice with 50 mM Tris buffer (pH 7.0). Washed. 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 result is shown in FIG. 3 (b).

In Figures 3 (a) and 3 (b), columns 1 and 4 represent Escherichia coli without plasmids, columns 2 and 5 represent E. coli with plasmids, and column 3 shows standard proteins. 43, 29, 18 and 14 kilodaltons are shown from above as molecular weight.

Through the present invention, E1E2 protein, which is an antigen-determining site fusion protein of the envelope gene of Korean hepatitis C virus, can be mass-produced in E. coli by genetic engineering method, and in particular, a fusion protein of the envelope protein fragments in which antibody binding sites are concentrated By using this method, it is possible to diagnose hepatitis C more precisely and more sensitively than the antibody diagnosis method using the protein obtained from the entire coat 1 and coat 2 genes, and contribute greatly to the development of a vaccine against HCV.

Claims (7)

A recombinant fusion protein comprising the antigenic determinant of the envelope protein of the Korean hepatitis C virus having the following amino acid sequence. The recombinant fusion protein of claim 1, further comprising ubiquitin. DNA comprising a nucleotide sequence encoding the protein of claim 1. The DNA of claim 3 comprising the following three nucleotide sequences encoding the epitope of the envelope protein of the Korean hepatitis C virus. An expression vector ptrpH-UB-E1E2 comprising the DNA of claim 3. E. coli transformed with the expression vector of claim 5. A method for producing the recombinant fusion protein of claim 1, comprising culturing E. coli of claim 6.