KR100361885B1 - Nucleotide sequences of korean hepatitis c virus cdna fragments and amino acid sequences deduced therefrom - Google Patents

Abstract

PURPOSE: Nucleotide sequences of Korean hepatitis C virus cDNA fragments and amino acid sequences deduced therefrom are provided, thereby applying them to studies on hepatitis C diagnosis. CONSTITUTION: Korean hepatitis C virus(KHCV) cDNAs have the nucleotide sequences of figures 1 to 8 and the KHCV polypeptides have the amino acid sequences of figures 1 to 8. A method for producing one KHCV polypeptide having the amino acid sequence of figures 1 to 8 comprises the steps of: constructing a vector containing KHCV cDNA having one nucleotide sequence of figures 1 to 8; transforming a microorganism with the vector; and culturing the transformed microorganism.

Description

Nucleotide Sequences and Amino Acid Sequences Inferred from the New Korean Hepatitis C Virus cDNA Fragments

The present invention relates to cDNA gene fragments and novel fragments of a new type of hepatitis C virus (HCV) obtained from a Korean non-A hepatitis (NANBH) patient. It relates to a polypeptide having an amino acid sequence encoded.

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 were identified in the 1980s, and diagnostic reagents and vaccines were 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 have cirrhosis. (Cirrhosis) and known as a terrible epidemic that spreads to Hepatocellular Carcinoma, it has only been found that human non-ABB 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 not been discovered until recently. It was. 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 the proteins (5-1-1, C100) produced by expressing the cloned partial gene fragments in E. coli and yeast were obtained from serum of hepatitis C patients. (Science 244, 362-364 (1989)), while 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)), and claimed that the base sequence was a subtype different from that of the C-type virus obtained after infection with chimpanzees.

The present inventors have performed immunoassay methods from hepatitis C virus cDNA libraries prepared from the sera of several Korean hepatitis C patients (Young, RA & Davis, RW, Proc. Natl. Acad. Sci. USA 80, 1194 (1983); Huynh, TV et al., In DNA Cloning; A Practical Approach, Vol. 1 (DM Glver, ed.) Pp 49-78, IRL Press, Oxford, UK (1985)). Successful identification of the unique sequences of hepatitis virus (KHCV-LSC1) cDNA fragments (Korean Patent Application No. 91-9510, filed on June 10, 1991).

On the other hand, Okamoto et al. (Okamoto et al., J. Jen, Virol, 73, 673-679 (1992)) devised a PCR primer specific for the type to protect the hepatitis C virus from four types (I, II, III). , IV) and HC-J6 (type III; Okamoto et al., J. Gen. Virol. 72, 2697-2704 (1991)) and HC-J8 (type) IV; Okamoto et al., Virology 188, 331-341 (1992).

Since the manufacture or diagnosis of a vaccine against hepatitis C virus uses a specific reaction between antigens and antibodies, it is necessary to use a protein produced by the gene of hepatitis C virus according to the characteristics of the region or country.

Based on these facts, the present inventors have made efforts to find a new type of hepatitis C virus in addition to the hepatitis C virus previously found in Korea. The cDNA fragment of another hepatitis C virus, which is significantly different from Korean Patent Application No. 91-9510, filed on the 10th, was cloned to determine its nucleotide sequences and to reveal the encoded amino acid sequence. It became.

An object of the present invention is to prepare cDNA fragments of a new type of Korean hepatitis C virus, determine its nucleotide sequence and reveal the amino acid sequence inferred therefrom, thereby making it possible to prepare an improved hepatitis C diagnostic reagent and vaccine. Hepatitis C virus cDNA fragments and polypeptides encoded therein are intended to contribute to the diagnosis and prevention of hepatitis C.

Hereinafter, the present invention will be described in detail.

First, the total RNA of HCV is extracted from the serum containing Korean hepatitis C virus by RNAzol B method (Chomczynski et al., Anal. Biochem. 162, 156-159 (1987)).

CDNA is prepared using a 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 is 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. In addition to the random primers mentioned above, cDNA may also be synthesized using antisense primers.

On the other hand, from the nucleotide sequence information of the cDNA fragments of the Korean type hepatitis C virus (see Korean Patent Application No. 92-10039, which is filed by the applicant), a relatively conserved portion of the nucleotide sequence is selected and several polymerases corresponding thereto are selected. Synthesizing the primer pair for the chain reaction. The primers for the polymerase chain reaction were synthesized by a DNA synthesizer (Applied Biosystems Inc. Model 380 B, USA) using automated solid phase phosphoamidite chemistry and electrophoresed on modified polyacrylamide gels. Then 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 1 minute; 55 ° C., 1 minute; Amplify the cDNA fragments by polymerase chain reaction with a temperature cycle program of 72 ° C., 3 minutes, repeated 40 to 45 times.

Each of these amplified cDNA fragments was separated by polyacrylamide gel electrophoresis to make blunt ends with the Klenow enzyme.

Smoothed terminal cDNAs were cloned into vector N13mp18 (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 et al., Proc. Natl. Acad. Sci. 74, 5463 (1977) (see FIGS. 1-8).

The determined nucleotide sequence showed 54% to 62% homogeneity as compared with the nucleotide sequence of KHCV-LBC1 (Korean Patent Application No. 91-9510, filed on June 10, 1991 by the applicant) (FIGS. Figure 16) showed a homogeneity between 85% and 92% between these.

The M13 phage group containing them (M13mp18-LBC-HCV3) was deposited with the ATCC on accession number ATCC 75449 as of April 21, 1993.

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) -4) unless otherwise specified.

Reference Example 1) Cleavage of DNA Using Restriction Enzyme

Restriction enzyme and reaction buffer were added to a reaction volume of 50 µl to 100 µl in a sterile 1.5 ml Eppendorf tube 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 Tris-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 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. 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 dithiothreitol, 10 mM ATP) , 0.5 mg / ml BSA) was used to perform the ligation reaction. Usually, 10 units of ligating enzyme was used in a volume of 20 μl, and 100 units of ligase was used for ligation of DNA having blunt ends. The reaction was carried out at 16 ° C. for at least 2 hours. The ligase was inactivated by heating at 占 폚 for 15 minutes.

Reference Example 4) E. coli transformation

E. coli host cell JM101 (ATCC 33876) was inoculated in 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 may reach 0.25 to 0.5. Incubated at 37 ° C until Cells were then separated by centrifugation (2,500 × G, 10 minutes), followed by washing with 50 ml of 0.1 M MgCl ₂ , followed by centrifugation (2,500 × G, 10 minutes), followed by 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 overnight at 37 ° C. After heat treatment, M13 vector DNA was subjected to 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 of 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) Preparation of RNA

Cells were lysed by adding 300 μl of RNAzol B (Cinna / Biotecx; P.O.Box 1421, Friendswood, Texas, U.S.A.) to 100 μl of serum of 40 hepatitis C patients. After the cells were lysed, 150 μl of chloroform was added, shaken vigorously for 15 seconds, stopped for 5 minutes on ice, 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 distilled water treated with 10 μl of diethylpyrocarbonate (DEPC) and immediately used to synthesize cDNA.

1-2) Preparation of cDNA

Using 3-4 μl of RNA obtained in Example 1-1 as a template for reverse transcriptase, cDNA was prepared using a cDNA synthesis kit (Cat. No. 8267SA) from BRL (PO Box 6009, Gaithersburg, MD 20877 USA). 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 ₂ 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 beta mercaptoethanol 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 ₂ , 15 mM MgCl ₂ , 5 mM dithiothreitol), 1.25 μl of 10 mM dNTP (dATP, dGTP, dTTP, dCTP), 1 μl DEPC-treated distilled water was added to a random starting body of 0.1 g / l and 1.0 μl of RNase inhibitor (1 U / μl, Promega, USA) so that the total volume was 25 μl, followed by stirring well. Make sure that the mold and primer are attached well. 1.25 μl of Superscript H-reverse transcriptase (18 U / μl; BRL) was added thereto, followed by reaction at 37 ° C. for 1 hour to synthesize the first cDNA helix.

40 cDNAs were synthesized by repeating the above procedure.

Example 2 cDNA Amplification of HCV by Polymerase Chain Reaction

2-1) Design of primer

Referring to the nucleotide sequence of the Korean hepatitis C virus (prior patent application No. 92-10039), a portion of the nucleotide sequence relatively conserved was selected to design a primer pair for PCR as follows. Above each primer pair is the sense primer and below is the antisense primer. The base sequence and position of the prepared primers are based on the base sequence of KHCV-LBCl described in the patent application No. 92-10039.

The base sequence of all primers has a directionality from the 5 'end to the 3' end, the first number in parentheses indicates the position of the first base at the 5 'end, and the last number indicates the position of the last base at the 3' end.

Thus, if the preceding number is greater than the latter number, it represents an antisense primer and vice versa. Among the bases constituting the primer, the remaining bases except for 4 to 5 bases at the 3 'end can be changed slightly (2 to 3 out of 10 bases), and the 5' end is used to change the recognition site of the restriction enzyme. You can add

The primers designed as described above were synthesized by a DNA synthesizer using an automated solid phase phosphoamidite chemistry (Applied Biosystems, Inc., USA, model 380 B), and then modified polyacrylamide gel (2M). Isolation was performed by electrophoresis using urea, 12% acrylamide, 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% of acetonitrile: water mixture, and purified pure. 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-C1 pH8.3, 500 mM KCl, 15 mM MgCl ₂ , 0.1% (w / v) gelatin), 10 μl of dNTP mixture (1.25 mM of dGTP, dATP, dTTP, and dCTP), 0.2 μg each of sense and antisense primers, 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., 1 minute; 55 ° C., 1 minute; 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 then centrifuged to obtain amplified double helix nucleic acid, and 20 μl of TE buffer (10 mM Tris-HCl). , pH7.5, 1mM EDTA) was used in the next experiment.

The cDNAs thus amplified were separated by polyacrylamide gel electrophoresis, respectively, and smooth ends were made with Klenow enzyme.

The smooth-terminal cDNAs were cloned into M13mp18, and nucleotide sequences were determined by Sanger's dideoxy method. 32 of 40 (80.0%) were hepatitis C virus cDNA fragments similar to KHCV-LBC1, and the remaining 8 were cDNA fragments of hepatitis C virus that were significantly different from KHCV-LBC1. Of the eight cDNA fragments, cDNAs amplified by primers 1311S and 1864A were named fragments 4F4, 4H19, 4H28, 4H34 and 4I3, respectively, and cDNAs amplified by initiators 2527S and 3209A were named fragments 6H19, 6H34 and 6I3, respectively. . Their nucleotide sequences and amino acid sequences deduced therefrom are shown in FIGS. These base sequences are shown in FIGS. 9-16 as compared to KHCV-LBC1 (Patent Application 91-9510, filed on June 10, 1991 by Applicant).

The present invention is expected to contribute to hepatitis C diagnostic research and medical research by revealing the partial nucleotide sequence and amino acid sequence of other types of Korean hepatitis C virus.

1 shows the nucleotide sequence of fragment 4F4 and the amino acid sequence deduced therefrom,

2 shows the nucleotide sequence of fragment 4H19 and the amino acid sequence deduced therefrom,

3 shows the nucleotide sequence of fragment 4H28 and the amino acid sequence deduced therefrom,

4 shows the nucleotide sequence of fragment 4H34 and the amino acid sequence deduced therefrom,

5 shows the nucleotide sequence of fragment 4I3 and the amino acid sequence deduced therefrom,

6 shows the nucleotide sequence of fragment 6H19 and the amino acid sequence deduced therefrom,

7 shows the nucleotide sequence of fragment 6H34 and the amino acid sequence deduced therefrom,

8 shows the nucleotide sequence of fragment 6I3 and the amino acid sequence deduced therefrom,

9 compares the nucleotide sequence of fragment 4F4 with the corresponding nucleotide sequence of KHCV-LBC1,

10 compares the nucleotide sequence of fragment 4H19 with the corresponding nucleotide sequence of KHCV-LBC1,

11 compares the nucleotide sequence of fragment 4H28 with the corresponding nucleotide sequence of KHCV-LBC1,

12 compares the nucleotide sequence of fragment 4H34 and the corresponding nucleotide sequence of KHCV-LBC1,

13 compares the nucleotide sequence of fragment 4I3 and the corresponding nucleotide sequence of KHCV-LBC1,

14 compares the nucleotide sequence of fragment 6H19 with the corresponding nucleotide sequence of KHCV-LBC1,

15 compares the nucleotide sequence of fragment 6H34 and the corresponding nucleotide sequence of KHCV-LBC1,

Figure 16 shows a comparison of the nucleotide sequence of fragment 6I3 with the corresponding sequence of KHCV-LBC1.

Claims (19)

Korean type hepatitis C virus (KHCV) cDNA having the nucleotide sequence of FIG. KHCV polypeptide having the amino acid sequence of FIG. KHCV cDNA having the nucleotide sequence of FIG. KHCV polypeptide having the amino acid sequence of FIG. KHCV cDNA having the nucleotide sequence of FIG. KHCV polypeptide having the amino acid sequence of FIG. KHCV cDNA having the nucleotide sequence of FIG. KHCV polypeptide having the amino acid sequence of FIG. KHCV cDNA having the nucleotide sequence of FIG. KHCV polypeptide having amino acid sequence of FIG. KHCV cDNA having the nucleotide sequence of FIG. KHCV polypeptide having the amino acid sequence of FIG. KHCV cDNA having the nucleotide sequence of FIG. KHCV polypeptide having amino acid sequence of FIG. KHCV cDNA having the nucleotide sequence of FIG. KHCV polypeptide having the amino acid sequence of FIG. A vector comprising a KHCV cDNA having the nucleotide sequence of any one of FIGS. A microorganism transformed with the vector of claim 17. A method of preparing a KHCV polypeptide having an amino acid sequence of any one of FIGS. 1 to 8 by culturing the microorganism of claim 18.