JP3058436B2 - Nucleic acid fragment encoding hepatitis C virus constituent polypeptide and use thereof - Google Patents

Description

The present invention relates to a gene fragment encoding a virus-constituting polypeptide of hepatitis C virus (hepatitis C virus), a hepatitis C virus-constituting polypeptide, and uses thereof. About the law.

Background of the Invention and Prior Art It has long been known that there are two types of viral hepatitis, hepatitis A (infectious hepatitis) and hepatitis B (serum hepatitis). This was mainly based on the difference in the route of infection, and it was confirmed that hepatitis A caused an epidemic by oral infection and hepatitis B was transmitted mainly through blood. The two causative viruses of hepatitis have already been isolated and identified, and hepatitis A virus is a 27 nm diameter RNA virus belonging to the picornavirus (Finestone, SM et al.,
Science 182 p.1026 (1973), on the other hand, hepatitis B virus was found to be a 42-nm-diameter enveloped DNA virus belonging to the hepadnavirus (Dan
e, OS, et al., Lancet, Ip. 695 (1970)). At present, immunoserologic diagnostic methods for these hepatitis viruses have been established, and preventive measures have been almost established.

As a definitive diagnosis method for these two hepatitis viruses has been established, the existence of non-A non-B hepatitis that does not belong to any of them has been revealed (Prince, AM, et al., Lance).
tI p.241 (1974)). Post-transfusion hepatitis was greatly reduced by the introduction of hepatitis B virus surface antigen (HBsAg) screening, but did not become zero. Moreover, there was no evidence of hepatitis A or B infection from hepatitis patients who developed it. Could not be obtained. For this reason, this hepatitis was generally called non-A non-B hepatitis. In Japan, this type of hepatitis accounts for about 50% of sporadic hepatitis and about 90% of post-transfusion hepatitis, and it is estimated that more than 50% of chronic hepatitis, cirrhosis, and liver cancer are caused by non-A non-B hepatitis. Has become a major social problem.

Separately, it has been found that there is a second viral non-A non-B hepatitis prevalent by oral infection, such as in India, Myanmar, Afghanistan, or North Africa (Khuroo, MSAm. J. Med. , 68 p. 818 (1980)), which is commonly referred to as waterborne or epidemic non-A non-B hepatitis. In Japan, this hepatitis epidemic has not been observed, but some imports of hepatitis from endemic areas by travelers seem to be observed (Fukuhara et al., Proceedings of the 25th Annual Meeting of the Japanese Society of Hepatology).

The present invention relates to the above-mentioned blood type non-A non-B hepatitis virus, which mainly infects via blood,
This virus is referred to herein as hepatitis C virus.

Regarding hepatitis C, the virus itself has not yet been isolated and identified, and thus, there is no established treatment or prevention method for this type of hepatitis. Conventionally, the diagnosis of hepatitis has been based on the exclusion diagnosis. That is, a test for hepatitis A and B, for which a diagnostic method has been established for the serum of the patient, performs a test for hepatitis, denies these hepatitis, and further exhibits herpes like symptoms as a part of systemic infection,
He was diagnosed as hepatitis C by denying the possibility of cytomegalo and Epstein-Barr virus infection and denying drug-induced or alcoholic hepatitis and autoimmune hepatitis.

The infectious nature of the hepatitis virus is 19
In 1978, it was proved by an American research group in infection experiments using chimpanzees (Tabor, E., et al., Lancet I
p.463 (1978)). However, despite much effort around the world, the pathogen virus remains unknown for more than a decade. Almost all approaches used in studies of hepatitis A and B, such as sedimentation reaction in agar gel, immunoelectron countercurrent method, radioimmunoassay, fluorescent antibody method, and electron microscope method, using blood and liver tissue of infected chimpanzee Have searched for viruses and related antigen-antibody systems, but have not yet obtained what is said to be certain.

It can be said that the history of hepatitis C virus investigation was a history of expectation and disappointment. Many viruses or antigen-antibody candidates have emerged, but they have been rejected one after another (Prince, AM, Ann. Rev. Microbiol., 37,
p.217 (1983)). A recent example is the retroviral theory of Seto et al. (Seto, B. et al., Lancet I p. 941 (198
4)) According to them, reverse transcriptase activity was detected in serum and blood products that have been shown to cause hepatitis C in chimpanzees. This enzyme was found to be 1.14 g / ml in sucrose density gradient centrifugation. , That is, it has a floating density similar to that of a retrovirus. Subsequently, Prince et al. Reported that retrovirus-like particles were observed by inoculating patient serum into chimpanzee primary liver cells (Prince, AM et al., Lancet I p. 1071 (198
Four)). However, reverse transcriptase activity was ruled out by Hollinger et al. (Hollinger et al., Lancet I p.41
(1986)). Furthermore, the virus particles observed by Prince et al. Were denied as contamination with myxovirus.

The problems that make the study of hepatitis C difficult are that the virus concentration in the serum is as low as 10 ² to 10 ^3, and that the presence of antibodies such as chimpanzees reinfected with the same inoculum is suspected, Infection experiments are limited to chimpanzees and marmosets.

Recently, reports of the capture of hepatitis C virus cDNA were reported by Chiron (Choo, Q et al., Science, 22nd, USA).
4, p.359 (1989); Ku., G. et al., Science, 224, p.362 (19
89)) and Okamoto et al. Of Jichi Medical University (Japan Society of Hepatology,
1990), and the full picture of this viral gene is being revealed.

By the way, in general, the difference between viruses is completely different depending on the difference in immunoserologic properties and the difference in molecular genetic properties. In addition, differences in strains cause some differences in immunoserologic properties, so that the same diagnostic method results in differences in detection sensitivity due to differences between strains, and in vaccines, differences in immunogenicity and infection protective ability. Molecular genetic diagnostic methods such as DN
In A probe diagnosis, it is generally known that hybridization between a probe and a viral nucleic acid is not practical unless the homology at the nucleic acid level is very high. In other words, it is expected that DNA hybridization will not occur due to differences at the nucleic acid level between strains, and DNA probe diagnosis cannot be effectively achieved.

Hepatitis B, which is well known and well analyzed as serotype hepatitis, is a major hepatitis B virus subtype in each region such as Europe, the United States, and Southeast Asia, that is, an endemic strain characteristic of that region. Is known to exist. Therefore, even in the hepatitis C virus targeted by the present invention, it is conceivable that a virus species or virus strain peculiar to a region exists.

Hitherto, as a reagent for measuring antibody to hepatitis C, a commercially available product developed by Chiron-Ortho, Inc. (Ortho HC
VAb ELISA test), which uses an antigen expressed in yeast as a fusion protein with hepatitis C virus-related specific peptide and SOD (human superoxide dismutase). However, it has been revealed that this assay kit cannot detect all sera of hepatitis C patients prevalent in Japan (Japanese Society of Hepatology 1990
Year). This result is thought to reflect the antigenic difference between strains prevalent in the United States and strains prevalent in Japan. Therefore, in order to establish a method for diagnosing and preventing hepatitis C virus prevalent in a specific region, for example, especially in Japan, it is necessary to catch a major hepatitis C virus strain in Japan.

Further, since the antibody detected by the above detection system usually takes 3 to 6 months after the onset of hepatitis C to become positive, the biggest problem is that it cannot be used as a diagnostic method for hepatitis C during this time. I am worried. In addition, even in cases where only negative blood is transfused in the detection system, the presence of hepatitis C virus is often clarified. Therefore, this antibody test alone can eliminate only about half of post-transfusion hepatitis. Thus, a new antibody test or a method for detecting a virus structural protein antigen is eagerly desired.
In addition, since the so-called C100 antigen used in the detection system is derived from a non-structural protein gene, the identification of a viral structural protein and its antigen-antibody detection system as a more direct diagnostic method or as a candidate for future vaccine materials are considered. Is very important.

Object of the invention Under such circumstances, the present inventors have conducted repeated studies for the purpose of cloning the pathogenic virus of hepatitis C or the gene of the virus. As a result, the present inventors derived hepatitis C virus from serum of hepatitis patients. We successfully cloned the gene encoding the antigenic peptide sequence.

In other words, the present inventors used heavier GPT plasma from blood donors and took advantage of immunoscreening methods incorporating new molecular genetic techniques, different from conventional immunoserologic methods, to reduce hepatitis C virus. When only the hydrophilic region of the unique gene fragment was amplified by the PCR method and the expression of this gene fragment in Escherichia coli was attempted, a peptide that reacted strongly with the serum of a hepatitis C patient was finally obtained. The present invention provides these gene fragments, polypeptides predicted therefrom, and uses thereof.

Structure and Effect of the Invention When cloning a nucleic acid fragment as the object of the present invention, a liver of a Japanese infected with hepatitis C or a liver of a chimpanzee infected with hepatitis C was used as a research material. One strategy is to extract and synthesize cDNA based on this, and select virus-specific cDNA from them by subtraction with chromosomal DNA. However, it is extremely difficult to secure a sufficient amount of good experimental materials necessary for this. As further research material, the plasma of hepatitis C infected persons or infected chimpanzees can be considered. The presence of a carrier of hepatitis C has been confirmed in humans, and the higher the donor's GPT level in transfusion, the higher the frequency of non-A non-B hepatitis after blood transfusion. Is estimated to be high. Therefore, the present inventors pooled GPT-high plasma from Japanese blood donors, which were available in relatively large quantities, and used them as research material. In addition, the plasma of a chimpanzee that has developed hepatitis by inoculating the serum of a Japanese hepatitis C patient can be used, but at present there are still some problems due to the difficulty of obtaining chimpanzees.

Hepatitis C virus concentration in plasma was 10
^Since it is estimated to be only about ² to 10 ^3, it is necessary to concentrate the virus about 1,000 times for extracting the viral nucleic acid and synthesizing the cDNA. However, human plasma is 7%
It is impossible to simply concentrate the protein solution before and after, and it is necessary to concentrate only the virus while removing the protein. The polyethylene glycol used by the present inventors (PE
The formation of a precipitate with a precipitant such as G) can be performed relatively easily, is suitable for treating a large amount of plasma, and is a mild method with little virus inactivation. Besides this,
Pelletization by ultracentrifugation, salting out in addition of salts such as ammonium sulfate, ultrafiltration, gel chromatography and the like can be used. The plasma concentrated about 1,000-fold as described above is treated with guanidium thiocyanate, extracted with phenol / chloroform, concentrated by ethanol precipitation, and the total nucleic acid in the plasma is purified. Then DNA
Decompose human-derived DNA that has been contaminated with a degrading enzyme, and perform RNA extraction by phenol / chloroform extraction and ethanol precipitation.
Is purified. CDNA is synthesized from the purified RNA and inserted into a λgt11 vector to prepare a cDNA library.

The λ phage is infected into E. coli, inoculated on a bacterial culture plate, and cultured at 42 ° C. for several hours. Then, cover with a nitrocellulose filter (NC filter) and incubate for several hours, peel off the NC filter and take a replica. This replica is treated with a blocking solution, washed with PBS or the like, and then subjected to immunoscreening. That is, the replica is reacted with human or chimpanzee serum in the recovery phase of hepatitis C or acute phase, washed with PBS or the like, reacted with enzyme-labeled anti-human IgG or IgM, washed, and reacted with a substrate solution to develop color. . Phages corresponding to the developed plaques were selected and subjected to a secondary screening to obtain reproducible clones.

These clones were subcloned, and a 0.9 Kbp insert (CE-5'JII) was confirmed by agarose gel electrophoresis. The nucleotide sequence of CE-5'J II was determined by the dideoxy method, and the results are shown in FIG. CE-5'J II
The amino acid sequence predicted from the nucleotide sequence of is shown in FIG. Further, analysis of the hydrophilic / hydrophobic region based on the above amino acid sequence based on the method of DOOLIT et al. Revealed that a peptide portion composed of 120 amino acids from the N-terminus contained 3 amino acids as shown in FIG. It was found that there were two strong hydrophilic regions. These hydrophilic regions seemed to be very significant from an immunological point of view. The present inventors amplify a gene fragment containing only these hydrophilic regions by PCR, and efficiently express this gene fragment as a fusion protein with β-galactosidase or directly alone in Escherichia coli. Confirmed. Western blots of the two types of expressed proteins thus obtained reacted strongly with the sera of hepatitis C patients, and were found to retain the hepatitis C virus antigenicity. The present invention not only discloses the nucleotide sequence of the gene fragment (CE-5'JII) specific to hepatitis C and the amino acid sequence predicted from the nucleotide sequence, but also discloses the antigen of hepatitis C virus. As a preferred example of a peptide having the property, a peptide consisting of amino acids of 120 hydrophilic regions as shown in FIG. 3 is also disclosed.

Recently, synthetic peptides have been used for the analysis of protein epitopes or the development of antibody measurement reagents, but this method can capture sequential epitopes, but does not provide epitopes for higher-order structures of peptides and proteins. Is considered difficult to capture. The protein obtained by the present invention is a novel protein suitable for use as a reagent for measuring hepatitis C-related antibody or a vaccine without including the above-mentioned problems. Further, the gene sequence itself obtained in the present invention is extremely useful for the development of a DNA probe diagnostic reagent for hepatitis C. The nucleic acid fragment encoding the hepatitis C virus constituent polypeptide of the present invention, the hepatitis C virus constituent polypeptide, and various methods for detecting hepatitis C virus using the same are particularly useful for the detection of hepatitis C virus in Japan. It is considered to be extremely useful in

In addition, the hepatitis C virus constituent polypeptide provided by the present invention is highly likely to be the core region of hepatitis C virus, as inferred from the gene structure of a closely related flavivirus. Therefore, the hepatitis C virus-constituting polypeptide of the present invention has a higher antigen-antibody activity than the non-structural region peptide compared to the hepatitis C virus non-structural region peptide used in a commercially available antibody detection system kit. System allows direct detection of hepatitis C virus.

Hereinafter, the present invention will be described in more detail with reference to Examples. However, these Examples illustrate various specific examples of the present invention, and the present invention is not limited to these Examples. Absent.

Example (1) Enrichment of GOT and GPT high human pool plasma High HPT antigen-free and GPT value of 100 provided by the Japanese Red Cross Society
The above human pooled plasma (approximately 8.2 liters) was
It was concentrated by a factor of 0. First, human pool plasma was subjected to rough centrifugation to remove insolubles. 1/10 volume of 5M sodium chloride solution,
Next, a 1/10 volume of a 40% (W / W) polyethylene glycol solution (PEG 6,000, manufactured by Wako Pure Chemical Industries, Ltd., average molecular weight 7,500) was added at 4 ° C. with stirring. After standing for 1 hour, the supernatant was removed by centrifugation at 7,000 rpm for 20 minutes, and a TNE solution (10 mM Tris-HCl, pH 7.4, 1 mM ED
TA, 140 mM NaCl) and redissolved. This solution was layered on top of a centrifuge tube in which 20%, 15%, 10% and 5% TNE solutions of sucrose were layered stepwise, and ultracentrifuged at 4 ° C. and 80,000 × G for 12 hours. After separation, the supernatant was removed and the sediment was washed with 8 ml of P.
It was dissolved in BS to make a 1,000-fold concentrate of GOT and GPT high human pool plasma.

(2) Purification of RNA from GOT and GPT high-pool human pool plasma concentrate First, 5 ml of a guanidium thiocyanate solution (4 M guanidium thiocyanate, 50 mM Tris-HCl pH7.6, 10mM EDTA, 0.1M 2
-Mercaptoethanol, 2% sarkosyl) was added, and the mixture was stirred, extracted with phenol / chloroform, and purified by ethanol precipitation using glycogen as a carrier to purify all nucleic acids in the concentrated plasma. Next, in order to degrade human-derived DNA present in the total nucleic acid, RNase-free DNase 1.1 was used in the presence of 2 mM vanadyl ribonucleotide complex.
5KU / ml (Boehringer / Mannheim), 50mM Tris
The mixture was treated at 37 ° C. for 30 minutes in 400 μ of a mixture of −HCl pH 7.4, 1 mM EDTA, and 10 mM MgCl ₂ . Then, 250 mM EDTA solution 1
The reaction was stopped by adding 6 μm and 8 μm of 10% SDS solution, and RNA was purified by phenol / chloroform extraction and ethanol precipitation. Further, in order to remove a large amount of glycogen present in the RNA and impurities considered to be present in a minute amount, a purification operation was performed using QIAGEN pack-100 (manufactured by QIAGEN).

(3) Construction of cDNA Library cDNA was synthesized from all of the RNAs purified by the above method using a cDNA synthesis system (Amersham).
Next, the synthesized cDNA was cloned into a λgt11 vector by cDNA cloning λgt11 (Amersham).
As a result of invitro packaging, a library of 1.2 × 10 ⁶ plaque forming units (PFU) was obtained.

(4) Screening of HC virus-related clones using chimpanzee sera during recovery and carrier phases of hepatitis C (HC) (A) Preparation of Escherichia coli lysate The primary antibody used for screening the cDNA library is chimpanzee during HC recovery phase and carrier phase Due to the plasma, a high non-specific reaction was expected. Therefore, in order to suppress this non-specific reaction, a lysate of Escherichia coli Y1090 used for the operation of absorbing chimpanzee plasma for screening was prepared. That is, 50 μg / ampicillin from a single colony
ml of LB medium (1% Bacto-tryptone (Difco), 0.5% Bacto-yeast extract (Difco), 1 ml
% NaCl, pH 7.5), 20 ml of E. coli Y1090 culture cultured overnight at 37 ° C was added to 2 LB medium, and further cultured at 37 ° C overnight. This culture solution was transferred to a centrifuge tube, and centrifuged at 4,000 ° C. for 10 minutes at 4 ° C., and the supernatant was removed.
RIPA solution (1% sodium deoxycholate, 1% Trit
on X-100, 0.3 M NaCl, 0.1% SDS, 0.1 M Tris-HCl p
H7.5, 1 mM PMSF) to solubilize it and further add 9,0
After centrifugation at 00 rpm for 10 minutes at 4 ° C., the supernatant was used as E. coli lysate.

(B) Preparation of a replica filter for antibody screening From a cDNA library constructed from RNA in GOT, GPT high-level human pool plasma concentrate, one LB plate [1.5%
Agar (Nissui Pharmaceutical), 1% Bacto-tryptone, 0.5%
Bacto-yeast extract, bacterial culture plate containing 1% NaCl pH 7.5, 50 μg / ml ampicillin (Nunc; 23c
m × 23 cm)] and take 10,000 PFU of phage per E. coli Y1
Infect 090 at 37 ° C for 15 minutes and use Top Agar 40ml (0.7% A
gar, 1% Bacto-tryptone, 0.5% Bacto-yeast extrac
t, 1% NaCl, pH 7.5, 50 μg / ml ampicillin) and cultured at 42 ° C. for 4 to 5 hours. Then, 10mM IPTG
Nitrocellulose filter (NC filter: S & S, Code BA85, 23cm × 23c) impregnated with (Sigma)
m), and the culture was further continued at 37 ° C. 3 hours later, N
The C filter was peeled off from the plate, washed with PBS, immersed in a blocking solution (PBS solution containing 5% skim milk, 0.05% NaN ₃ ) and shaken at 4 ° C overnight.

(C) Antibody screening Replica filter soaked overnight in blocking solution
After washing with PBS, chimpanzee pool plasma in HC recovery phase and carrier phase diluted 10-fold in PBS (plasma for screening; chimpanzee pool plasma in HC recovery phase and carrier phase was diluted 5-fold in PBS, Add E. coli lysate and perform the non-specific reaction absorption operation at 4 ° C overnight.
(Diluted twice with PBS) and reacted while shaking at room temperature. After 2 hours, the replica filter was washed with PBS-T (PBS solution containing 0.05% Tween 20) for 15 minutes each time, for a total of 3 times, and then a 1,000-fold diluted peroxidase-labeled anti-human IgG and IgM goat antibody (MBL) Company, Fa
It was immersed in an incubation buffer (b) containing PBS (1% bovine serum albumin) and reacted at 37 ° C. with shaking. After 1 hour, the plate was washed with PBS-T for 15 minutes each time, 4 times in total, and then washed with PBS for 5 minutes, and then a color developing solution (0.02% DAB (Sigma), 0.1% NaCl ₂ .6H ₂ O, 0.005%)
% H ₂ O ₂ ) for color development. Phages corresponding to the plaques developed on the NC filter were selected and subjected to secondary screening. That is, each phage 200PFU selected in the primary screening was separately phage-free
Escherichia coli Y1090 was infected together with 200 PFU, and seeded on an LB plate of a 90 mm petri dish (manufactured by Becton Dickinson) to prepare a replica filter. These were subjected to antibody screening by the above-mentioned method, and one clone of phage which reacted with the chimpanzee plasma in the HC recovery phase or the carrier phase with good reproducibility was obtained. The phage DNA can be prepared by a standard method (experimental medicine
Extra Number, Genetic Engineering Summary 5 (11), p.31 (198
7)) and digested with the restriction enzyme EcoR I (manufactured by Toyobo), and then digested with the pBluescript II SK (-) vector EcoR I.
Inserted into the I site and subcloned (Hanaha
n, D., J. Mol. Biol. 166, p557 (1983)). After the subcloned plasmid pCE-T3 was digested with EcoRI and electrophoresed on a 2% agarose gel, about 0.9 Kbp was obtained.
Was confirmed (CE-5'J II).

(5) Nucleotide base sequence and amino acid sequence of CE-5'J II (A) Determination of base sequence of CE-5'J II clone Plasmid containing CE-5'J II gene fragment
DNA was used as a template and used for [α- ₃₂ P] dCTP (800 Ci / mmol) reaction. The polymerase reaction with Klenow fragment was performed using Takara Shuzo's 7DEAZA sequencing kit. Using 8% polyacrylamide-8M urea gel, the mixture was electrophoresed at 1,800 V for 4 hours and exposed for 16 hours.

(B) Obtained base sequence and predicted amino acid sequence The base sequence obtained as a result of the above and the result of decoding the predicted amino acid sequence are shown in FIGS. 1 and 2, respectively. In addition, the hydrophilicity / hydrophobicity profile of the predicted amino acid sequence of CE-5'JII was analyzed based on the method of DOOLIT et al. FIG. 3 shows the results.

(6) Construction of Expression Plasmid PCR was performed to obtain a 360 bp (120 amino acids) gene fragment from the 5 'side of the CE-5'JII gene fragment. Using the CE-5'JII gene fragment as a template, 10 mM Tris-
HCl pH 8.3, 50 mM KCl, 1.5 mM MgCl ₂ , 0.01% (w / v) gelatin, 100 nM dNTp, 250 nM primer (the base sequence is shown in FIG. 4), 50 μM of 20 U / ml Taq Polymerase
The reaction was carried out for 35 cycles at 94 ° C. for 30 seconds, 55 ° C. for 30 seconds, 72 ° C. for 1 minute (a thermal cycler manufactured by Perkin-Elmer Cetus) was used. After extracting and purifying the gene fragment (JCC) amplified by PCR with phenol / chloroform, this was digested with the restriction enzymes Nco I and Hind III.
To expose the restriction enzyme cleavage sites. On the other hand, plasmid pKK233-2 (Pharmacia) was cut with restriction enzymes NcoI and HindIII, and then extracted and purified with phenol / chloroform. The thus obtained two gene fragments, 100 ng each, were mixed with a T4 ligase reaction solution (66 mM Tris-HCl pH 7.6, 6.6 mM MgCl ₂ , 10 mM DDT,
(1 mM ATP) using 2 units of T4 ligase at 4 ° C. for 1 hour. Yasutaka Takagi ed., "Genetic manipulation experiments" p161
E. coli JM109 was transformed with this reaction solution, and a plasmid was prepared from a colony growing on an agar medium containing 50 μg / ml of ampicillin (Metabolism Vol. 17, Chapter 4, “Lipase”, p81 (1980)). reference). as a result. p
A plasmid pKJCC4 in which a JCC gene fragment was inserted between NcoI and HindIII of KK233-2 was obtained. In the same manner, a plasmid pUEJCC1 in which a JCC gene fragment was inserted between EcoR I and BamHI of plasmid pUEX2 was obtained (see FIG. 5).

(7) Expression of ICC protein in Escherichia coli Escherichia coli having the plasmid pKJCC4 was inoculated in 5 ml of LB medium (0.5% yeast extract, 1% bactotryptone, 0.5% NaCl) and cultured at 37 ° C. overnight. Then, 5 ml of LB medium containing 2.5 mg of IPTG (Takara Shuzo) was added, and the cells were further cultured at 37 ° C. for 4 hours. After collecting the culture by centrifugation, 1m
TBST containing M PMSF (50 mM Tris-HCl pH 7.9, 150 m
M. NaCl, 0.5% Tween 20), the bacterial pair was suspended, 1 g of glass beads was added thereto, and the cells were destroyed by vigorously stirring at 4 ° C. for 15 minutes. After separating the cell disruption liquid and the glass beads, an insoluble fraction was obtained by centrifugation.
This was mixed with 1 ml of TE (50 mM Tris-HCl pH 7.5, 10 mM EDTA).
The suspension was examined for the presence or absence of antigen production by the following SDS-PAGE and Western blotting. Samples were electrophoresed on a 17% SDS-polyacrylamide gel and stained with 0.25% CDD. Similarly, the electrophoresed sample was electrically transferred to a nitrocellulose filter, and the filter was reacted with 5% skim milk for 1 hour at room temperature, and reacted with hepatitis C patient serum for 1 hour. After washing the filter with TBST, it was reacted with an anti-human IgG peroxidase-conjugated antibody (manufactured by Bio-Rad) for 1 hour at room temperature. TBST
And washed with hydrogen peroxide and diaminobenzidine. As a result, a band having a molecular weight of about 15,000 was observed in the E. coli extract solution sample having pKJCC4. This band almost coincided with the molecular weight estimated from the JCC gene fragment of the present invention. On the other hand, no band reacting with the serum of a hepatitis C patient was observed in a sample of an E. coli extract containing only pKK233-2, which is a negative control (see FIG. 6).

(8) Expression of βgal-JCC in E. coli E. coli containing plasmid pUEJCC1 was added to 10 ml of LB medium (0.5%
Yeast extract, 1% bactotryptone, 0.5%, NaCl) was inoculated, cultivated at 30 ° C. overnight, adjusted to a turbidity of 80 with a Kretmeter, and cultured at 30 ° C. for 90 minutes. Incubation temperature 42 ℃
After culturing for another 2 hours, the cells were collected by centrifugation. TBST containing 1 mM PMSF (50 mM Tris-HCl pH 7.9, 15
The cells were suspended in 0 mM NaCl, 0.5% Tween 20), 1 g of glass beads were added thereto, and the cells were disrupted by vigorously stirring at 4 ° C. for 15 minutes. After separating the cell disruption liquid and the glass beads, an insoluble fraction was obtained by centrifugation. This was suspended in 1 ml of TE (50 mM Tris-HCl pH7.5, 10 mM EDTA), and the suspension was subjected to SDS-PAGE (8
% Gel), and the presence or absence of antigen production was examined by Western blotting. As a result, in the sample of the E. coli extract containing pUEJCC1, one broad hand having a molecular weight of about 131,000 was observed. This band almost coincided with the molecular weight estimated from the JCC gene fragment of the present invention. On the other hand, it is a negative subject
A band reacting with the serum of a hepatitis C patient was not observed in a sample of an E. coli extract containing only pUEX2 containing no JCC gene (see FIG. 6).

[Brief description of the drawings]

FIG. 1 shows CE-5'J cloned according to the present invention.
2 shows the nucleotide sequence of II. FIG. 2 shows the amino acid sequence predicted from CE-5'J II. FIG. 3 shows the hydrophilic / hydrophobic profile of the peptide having the amino acid sequence shown in FIG. FIG. 4 shows the nucleotide sequences of the primers used for PCR. FIG. 5 shows plasmids pKJCC4 and pUEJCC1. FIG. 6 shows the results of SDS-PAGE and Western blot of the expressed protein.

Continuation of the front page (51) Int.Cl. ⁷ Identification symbol FI C12Q 1/70 C12Q 1/70 (C12P 21/02 C12R 1:19) Examiner Mayumi Saito (56) References JP-A-4-30791 (JP) JP-A-4-45795 (JP, A) JP-T2-500880 (JP, A) JP-T4-504715 (JP, A) JP-T5-506230 (JP, A) International publication 91 / 1376 (WO, A1) The Japanese Journal of Experimental Medicine, Vol. 60, No. 3 (June 1990), p. 167- 177 (58) Fields investigated (Int.Cl. ⁷ , DB name) C12N 15/00-15/90 C07K 14/00-16/46 G01N 33/50-33/98 C12P 21/00-21 / 08 C12Q 1/00-1/70 BIOSIS (DIALOG) GenBank / EMBL / DDBJ (GENETYX) MEDLINE (STN) WPI (DIALOG)

Claims (7)

(57) [Claims] 1. A nucleic acid fragment comprising a nucleotide sequence encoding a hepatitis C virus constituent polypeptide, wherein the nucleic acid fragment has the following nucleotide sequence, a sequence complementary to the nucleotide sequence, or 1 or 2 nucleotides in the nucleotide sequence. The nucleic acid fragment described above, comprising a base sequence in which several bases are deleted, substituted, or added. 2. A nucleic acid fragment comprising a nucleotide sequence encoding a hepatitis C virus constituent polypeptide, wherein said nucleic acid fragment comprises the following nucleotide sequence or a sequence complementary to said nucleotide sequence. Nucleic acid fragments. 3. A polypeptide comprising a hepatitis C virus-constituting polypeptide, wherein the hepatitis C virus-constituting polyplasmid has the following amino acid sequence or one or several amino acids deleted, substituted or substituted in the amino acid sequence: The above polypeptide, which comprises an added amino acid sequence. 4. A polypeptide comprising the following amino acid sequence or a hepatitis C virus constituent polypeptide consisting of at least 120 amino acids from the N-terminal side in the amino acid sequence. 5. A nucleic acid fragment according to the above (1) or (2), or a nucleic acid fragment comprising at least 360 bp from the 5 'side in the nucleotide sequence of the nucleic acid fragment, is incorporated into an appropriate expression vector, A polypeptide comprising the hepatitis C virus constituent polypeptide comprising the amino acid sequence according to the above (3) or (4), which is obtained by expressing it in a host cell. 6. A method for immunologically detecting a hepatitis C virus-related antibody, comprising using the peptide according to any one of the above (3) to (5). 7. The method according to claim 6, wherein the immunological detection method is selected from the group consisting of ELISA (enzyme-linked immunosorbent assay), RIA (radioimmunoassay), western blotting, and agglutination. Detection method.