JP3042864B2 - Non-A non-B hepatitis virus antigen peptide, nucleic acid fragment encoding the same, and use thereof - Google Patents

Description

The present invention relates to a serotype hepatitis virus (non-A non-B hepatitis virus) antigen peptide that is neither type A nor type B, a gene fragment encoding the same, and a fragment thereof. Regarding usage.

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 infection route, and it was confirmed that hepatitis A caused an epidemic by oral infection and hepatitis B was transmitted mainly through blood. The two hepatitis-causing viruses have already been isolated and identified. Hepatitis A virus is a 27 nm diameter RNA virus belonging to the picornavirus [Fineston, SMet
al., Science 182 p1026 (1973)], whereas hepatitis B virus was found to be a 42-nm-diameter enveloped DNA virus belonging to the hepadnavirus.
[Dane, OS, et al., Lancet, Ip695 (1970)] At present, immunoserodiagnostic methods for these hepatitis viruses have been established.

With the establishment of a definitive diagnosis method for these two hepatitis viruses, the existence of non-A non-B hepatitis that does not belong to any of them has been revealed [Prince, AM, et al., Lancet.
I p241 (1974)].

Hepatitis after blood transfusion is hepatitis B virus surface antigen (HBsAg)
Was significantly reduced by the introduction of the screening method, but did not reach zero.
No evidence of hepatitis B or B infection was obtained. For this reason, this hepatitis is generally called non-A non-B hepatitis.

This hepatitis accounts for about 50% of sporadic hepatitis and more than 90% of post-transfusion hepatitis in Japan, as well as chronic hepatitis, cirrhosis and liver cancer.
It is estimated that 50% or more is caused by non-A, non-B hepatitis, which is a major social problem.

Separately, it has been found that there is a second viral non-A non-B hepatitis prevalent by oral infection in India, Burma, Afghanistan, or North Africa [Khuroo, MSAm. J. Med. ., 68 p818-824, (1980)]. This is commonly referred to as aqueous or epidemic non-A non-B hepatitis. In Japan, this hepatitis epidemic has not been observed, but it seems that some imports of hepatitis from endemic areas of travelers are seen [Fukuhara et al., Proceedings of the 25th Annual Meeting of the Japanese Society of Hepatology, 151 pages (1989)] ].

The present invention relates to the former serotype non-A non-B hepatitis virus, which is mainly transmitted via blood, and is referred to as a non-A non-B hepatitis virus in the present specification. .

Regarding this non-A non-B hepatitis, the virus itself has not been isolated and identified, and therefore, a method for diagnosing, treating and preventing this hepatitis has not been established. The diagnosis of hepatitis was based on the exclusion diagnosis. That is, the patient's serum is tested for hepatitis A and B, for which a diagnostic method has been established, to deny these hepatitis, and to further show hepatic inflammation as a partial symptom of systemic infection. Herpes,
He denied the possibility of cytomegalo and Epstein-Barr virus infection, denied drug-induced, alcoholic, and autoimmune hepatitis and was diagnosed with non-A, non-B hepatitis.

In 1978, the virus responsible for this hepatitis was infectious.
In an experiment conducted by an American research group in Chimpanzee-based infection experiments [Tabor, E., et al., Lancet.
463 (1978)]. But despite much effort around the world, more than a decade later, the true nature of the causative virus remains unknown. From blood and liver tissue of chimpanzees infected with patients, almost all types of hepatitis A and B used in agar gel sedimentation, immunoelectron countercurrent, radioimmunoassay, immunofluorescence, electron microscopy, etc. The approach has been to search for viruses and related antigen-antibody systems, but nothing has been confirmed yet.

It can be said that the history of non-A non-B hepatitis virus research was a history of expectations and disappointments. A number of virus or antigen-antibody candidates have emerged, which have been rejected one after another [Prince, AM, Ann. Rev. Microbio
l., 37, p217, (1983)].

A recent example is the retroviral theory of Seto et al. [Se
to, B. et al .: Lancet. I p941-943 (1984)], they show that serum and blood products that have been shown to cause non-A, non-B hepatitis in chimpanzees have reverse transcriptase activity. It was detected and in sucrose gradient centrifugation the enzyme came to 1.14 g / ml, that is, it had a flotation density similar to retrovirus. Subsequently, Prince and colleagues reported that retrovirus-like particles were seen when chimpanzee primary liver cells were inoculated with patient serum [Prince, AM et al. L.
ancet. I: p1071-1075 (1984)]. However, reverse transcriptase activity was ruled out by Hollinger et al. [Hol
linger et al., Lancet, I p41 (1986)]. Furthermore, Prince
The observed virus particles were denied as contamination with myxovirus.

Problems that make research on non-A, non-B hepatitis difficult include the presence of antibodies, such as low serum virus concentrations of 10 ² to 10 ³ and chimpanzees that have re-infected with the same inoculum. There is doubt, and there are only chimpanzees and marmosets in experimental models.

Recently, there has been a report that Chiron of the United States has captured cDNA for non-A, non-B hepatitis virus [Choo, Qe.
t al., Science, 244, p359-362 (1989), Kuo, G. et al., S
cience, 244, p362-364 (1989)], but a part of its nucleotide sequence has been published [European Patent EP 318216 A], but the properties of the virus itself and the properties of the virus-constituting proteins have not been elucidated yet. .

In general, the difference in virus is completely different from the diagnosis method due to the difference in immunoserological properties and the difference in molecular genetic properties. In addition, differences in strains result in differences in detection sensitivity due to differences between strains in the same diagnostic method, and differences in immunogenicity and infectious protection in vaccines due to differences between strains in the same diagnostic method. It is generally known that in molecular genetic diagnosis methods, for example, DNA probe diagnosis, hybridization between a probe and a viral nucleic acid is not practical unless the homology at the nucleic acid level is very high. That is, due to differences at the nucleic acid level between strains,
There may be cases where DNA hybridization does not occur and DNA probe diagnosis cannot be performed effectively.

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 epidemic strain characteristic of the region ( Since it is known that a subtype exists, the non-A, non-B hepatitis virus targeted by the present invention may have a virus species or a virus strain peculiar to a region.

Therefore, it is necessary to catch a major non-A non-B hepatitis virus strain in Japan in order to establish a method for diagnosing and preventing non-A non-B hepatitis virus prevalent in a specific area, for example, in Japan. .

Object of the Invention Under such circumstances, the present inventors have conducted repeated studies for the purpose of cloning the non-A non-B hepatitis causative virus or its viral gene. The gene encoding the antigenic peptide sequence of hepatitis virus was cloned, and it was confirmed that the peptide encoded thereby specifically reacted with non-A non-B hepatitis patient serum.

That is, the present inventors have obtained a non-A non-B type obtained by immunoscreening method using a new molecular genetic technique which is different from the conventional immunoserologic method using GPT-high plasma of a blood donor. An object of the present invention is to provide a peptide unique to hepatitis virus, a gene fragment encoding the same, and a method for using the same.

When cloning a nucleic acid fragment as the object of the present invention, a Japanese liver infected with non-A non-B hepatitis and a chimpanzee infected with non-A non-B hepatitis were used as research materials. It is conceivable that mRNA is extracted and cDNA is synthesized using the liver of the above, and virus-specific cDNA is selected therefrom by subtraction with chromosomal DNA. However, it is extremely difficult to secure a sufficient amount of good experimental materials necessary for this.

As another research material, plasma of a non-A non-B hepatitis infected person or an infected chimpanzee can be considered. In humans, the presence of a carrier of non-A non-B hepatitis has been confirmed, and the higher the GPT value of a blood donor during transfusion, the higher the frequency of non-A non-B hepatitis after blood transfusion. It is estimated to be frequent. Therefore, we pooled relatively high-available GPT-high plasma from Japanese blood donors for study. In addition, plasma of a chimpanzee that has developed non-A non-B hepatitis by inoculating the serum of a Japanese non-A non-B hepatitis patient can be used, but at present some problems remain due to the availability of chimpanzees.

Since the non-A non-B hepatitis virus concentration in plasma is estimated to be only about 10 ² to 10 ³ as described above,
Extraction of viral nucleic acid and synthesis of cDNA require concentration of the virus about 1000 times. However, human plasma is a protein solution of about 7%, and cannot be simply concentrated. It is necessary to concentrate the virus while deproteinizing. The polyethylene glycol we used (PE
Precipitation with a precipitant such as G) is a mild method that can be performed relatively easily, is suitable for treating large amounts of plasma, and has little virus inactivation. In addition, pelleting by ultracentrifugation, salting out by adding salts such as ammonium sulfate, ultrafiltration, gel chromatography and the like can be used.

The plasma thus concentrated about 1000-fold is treated with guanidium thiocyanate, extracted with phenol / chloroform, and the total nucleic acid in the concentrated plasma is purified by ethanol precipitation. Next, the contaminating DNA derived from humans is degraded with a DNase, and RNA is purified by phenol / chloroform extraction and ethanol precipitation.

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, spread on a bacterial culture plate, and cultured at 42 ° C. for several hours. After this, cover with a nitrocellulose filter (NC filter) and incubate for several hours.
Remove the 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 allowed to react with a non-A non-B hepatitis recovery phase or acute phase human or chimpanzee serum, and after washing with PBS or the like,
It is reacted with an 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 secondary screening to obtain reproducible clones.

This clone was examined for non-A, non-B hepatitis specificity.

Plaque immunoassay was performed using chimpanzee IgG in the non-A, non-B hepatitis recovery phase, carrier phase, and normal phase. As a result, a clone highly specific to the non-A, non-B hepatitis carrier phase was obtained. This clone was subcloned, and an insert (CH-15) of about 380 bp was confirmed by agarose gel electrophoresis.

After purifying chromosomal DNA from chimpanzee normal and non-A non-B acute hepatitis liver and normal human leukocytes, performing agarose electrophoresis, Southern hybridization was performed using a ³² P-labeled CH-15 clone. went.
CH-15 does not react with any DNA and therefore CH-15
Turned out not to be chromosomal DNA.

The DNA sequence of the CH-15 clone of the present invention was determined by the dideoxy method. As a result, the CH-15 clone was non-A non-B
It was a cDNA fragment of a total of 372 bp derived from the hepatitis hepatitis virus gene, and its nucleotide sequence was as shown in FIG. This base sequence and the amino acid sequence deduced from this are databased (Genetyx-CD software development 1989)
The search found no viruses, bacteria or other homology known to date. further,
As a result of comparison with the nucleotide sequence and amino acid sequence of non-A non-B hepatitis virus (HCV) published by Chiron, USA, the nucleotide sequence of CH-15 was 83.1%, and the amino acid sequence was 89.5%.
A region with% homology was present in the Chiron published sequence. From these results, it was estimated that this clone was the same type as that cloned by Chiron Co., and was a non-A non-B hepatitis virus of a different strain.

Furthermore, from the amino acid sequence encoded by CH-15,
The following two peptides having different lengths (29mer and 37me
When r) was synthesized and reacted with non-A non-B hepatitis patient serum, it was confirmed that it reacted very specifically with non-A non-B hepatitis patient serum.

That is, the present invention discloses an antigen epitope with high specificity among the amino acid sequences encoded by CH-15.

Among the above peptides, particularly in the measurement of non-A non-B hepatitis virus antibody using a 37-mer, extremely excellent results in both specificity and sensitivity were obtained, even when compared with a similar measurement method using a 29-mer. Indicated.

The gene sequence of the present invention can be expressed using a known suitable expression system, or the non-A non-B hepatitis virus antigen peptide of the present invention can be obtained using a known chemical synthesis method. . As a known suitable expression system, any of prokaryotic and eukaryotic cell expression systems can be used, and if necessary, a fusion protein with an appropriate peptide can be used. On the other hand, a chemical synthesis method is convenient for preparing a relatively short peptide consisting of a specific epitope. Such a peptide of the present invention can be used for an antibody test for non-A non-B hepatitis virus, or an animal can be immunized with the peptide to produce an antibody specific to the peptide, and Non-A non-B
It is also possible to detect non-A non-B hepatitis virus in the liver tissue of hepatitis-infected patients.

Furthermore, the non-A non-B hepatitis virus obtained in the present invention is considered to be extremely useful for preparing a vaccine for preventing infection.

In addition, the gene sequence itself is non-A non-B hepatitis DNA
It is extremely useful for the development of probe diagnostic kits.

Such non-A non-B hepatitis virus antigenic peptides of the present invention, nucleic acid fragments encoding the same, and various detection methods for non-A non-B hepatitis viruses using these,
In particular, it is considered to be extremely useful in detecting non-A non-B hepatitis virus in Japan.

 Hereinafter, the present invention will be described in more detail with reference to examples.

Example (1) Concentration of human pool plasma with high GOT and GPT levels HBs antigen-negative and GPT value of 10 provided by the Japanese Red Cross Society
Zero or more human pooled plasma (8.5) was concentrated 1000-fold by the following method. First, human pool plasma was subjected to rough centrifugation to remove insolubles. 1/10 volume of 5M sodium chloride solution, then 1/10 volume of 40% (W / W) polyethylene glycol solution (PEG
6000, manufactured by Wako Pure Chemical Industries, Ltd., average molecular weight 7500) was added at 4 ° C. with stirring. After standing for one hour, 7000 rotations, 20
Centrifuge for 1 minute to remove the supernatant, and add about 1/20 of the original plasma to the sediment.
Volume of TNE solution (10 mM Tris-HCl, pH 7.4, 1 mM EDTA, 140 mM
NaCl) was added and redissolved. 20% of sucrose,
15%, 10% and 5% TNE solutions were layered on top of the stepped centrifuge tubes and ultracentrifuged at 80,000 × G at 4 ° C. for 12 hours. After separation, the supernatant was removed, and the precipitate was dissolved in 8 ml of PBS to obtain a 1000-fold concentrate of GOT and GPT high-value human pool plasma.

(2) Purification of RNA from GOT and GPT-rich human pool plasma concentrate First, 8 ml of the above-mentioned 1000-fold concentrated plasma was added with a 5-fold amount of a guanidium thiocyanate solution (4 M guanidium thiocyanate, 50 mM Tris-HCl pH7.6, 10 mM EDTA, 0.1 M 2-mercaptoethanol, 2% sarkosyl) was added, and the mixture was stirred and extracted with phenol / chloroform. The whole nucleic acid in the concentrated plasma was purified by ethanol precipitation using glycogen as a carrier. Next, in order to degrade human-derived DNA present in the total nucleic acid, in the presence of 2 mM vanadyl ribonucleotide complex, RNase-free DNase 1.15 KU / ml
(Boehringer / Mannheim), 50 mM Tris-HCl p
In a mixture of H7.4, 1mM EDTA, 10mM MgCl ₂ in 400μ, 37
It processed at 30 degreeC for 30 minutes. Then, 250 mM EDTA solution 16μ, 10
The reaction was stopped by adding 8 μ% of SDS solution, and RNA was purified by phenol / chloroform extraction and ethanol precipitation.
Furthermore, in order to remove a large amount of glucogen and a trace amount of impurities present in this RNA, QIAGEN
Purification was performed using pack-100 (manufactured by DIAGEN).

(3) Construction of cDNA Library All the RNAs purified by the above-described method were subjected to cDNA synthesis using a cDNA synthesis system plus (manufactured by Amersham). Next, the synthesized cDNA was subjected to cDNA cloning λgt11
(Amersham) to clone into the λgt11 vector. As a result of in vitro packaging, a library of 1.8 × 10 ⁶ plaque forming units (PFU) was obtained.

(4) Screening of NANBH virus-related clones using chimpanzee serum in the non-A non-B hepatitis (NANBH) recovery phase and carrier phase (A) Preparation of Escherichia coli lysate The primary antibody using cDNA library screening is NANBH recovery phase and carrier phase , 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, ampicillin 50
LB medium containing 1 μg / ml [1% Bacto-trytone (Difco), 0.5% Bacto-yeast extract (Difco), 1
% NaCl, pH 7.5], 20 ml of Escherichia coli Y1090 culture cultured overnight at 37 ° C was added to 2 LB medium, and further cultured at 37 ° C overnight. Transfer this culture to a centrifuge tube, 9000 rpm, 10 minutes,
After centrifugation at 4 ° C., the supernatant was removed to obtain a precipitate. 4 g of RIPA solution (1% sodium deoxycholate, 1% Triton X-100, 0.3 M NaCl, 0.1% SDS, 0.1 MT / g of this sediment)
ris-HCl (pH 7.5, 1 mM PMSF) was added to solubilize the mixture, which was further centrifuged at 9000 rpm for 10 minutes at 4 ° C., and the supernatant was lysed in E. coli.

(B) Preparation of replica filter for antibody screening From a cDNA library constructed from RNA in GOT and GPT high-level human pool plasma concentrate, one LB plate [1.5% A
gar (manufactured by Nissui Pharmaceutical Co., Ltd.), 1% Bacto-tryptone, 0.5% Bac
Bacterial culture plate containing to-yeast extract, 1% NaCl pH7.5, 50 μg / ml ampicillin (Nunc; 23 cm
× 23cm)] and take 10000 PFU of phage per E. coli Y109
0 at 37 ° C for 15 minutes, and Top Agar 40 ml (0.7% Aga
r, 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 × 23) impregnated with (Sigma)
cm), and the culture was further continued at 37 ° C. NC after 3 hours
Remove the filter from the plate, wash with PBS, blockin
g 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 NANBH recovery phase and carrier phase diluted 10-fold in PBS (plasma for screening) [chimpanzee pool plasma in NANBH recovery phase and carrier phase was diluted 5-fold in PBS to 1/20 volume Was lysed at 4 ° C. overnight, followed by two-fold dilution with PBS. And shaking at room temperature to react. Two hours later, PBS-T (0.05% Tween
After washing the replica filter three times with a PBS solution containing en20 for a total of 15 minutes each time, a 1000-fold diluted peroxidase-labeled anti-human IgG and IgM goat antibody (MBL, F
Ab) was immersed in an incubation buffer (PBS solution containing 1% bovine serum albumin) and reacted at 37 ° C. with shaking. After 1 hour, 15 times each time with PBS-T
After washing 4 times in total for 5 minutes and then in PBS for 5 minutes, the coloring solution [0.0
2% DAB _{(Sigma), 0.1% NiCl 2 · 6H} 2 O, 0.005% H 2 O
₂ ] to form a color. Phages corresponding to 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 infected with Escherichia coli Y1090 together with the phage 200PFU having no inserted fragment, and spread again 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 (CH-15) was obtained which reproducibly reacted with chimpanzee plasma in the recovery phase and the carrier phase of the NANBH.

(5) of each clone using antibodies in chimpanzee serum
Examination of specificity for NANBH For the clone obtained in (4), a replica filter was prepared in the same manner as in the secondary screening of (4) .C.
A plaque assay was performed using an H-recovery phase, a carrier phase and normal chimpanzee serum or an IgG fraction purified by DEAE-self wine column (manufactured by Seikagaku Corporation) after precipitation with ammonium sulfate. The method is the same as in the case of antibody screening, but when using the chimpanzee IgG fraction for the primary antibody reaction, dilute with PBS to a concentration of 50 μg / ml, add 1/20 volume of E. coli lysate, and add Was used overnight after absorbing the non-specific reaction.

As a result of the plaque assay, CH-15 reacted at a high rate with the chimpanzee serum or IgG fraction in the NANBH carrier phase, and did not react with the serum or IgG fraction of normal chimpanzee at all.

From these results, it can be said that CH-15 is a clone having high specificity for chimpanzee serum in the NANBH carrier phase.

The phage DNA of CH-15 was purified [see Special Issue of Experimental Medicine, Special Edition of Genetic Engineering, Vol. 5 (11), p. 31-32 (1987)]. Site and subcloned [Douglas Hanahan, J. Mol. Biol. 166 , P557-580 (198
3)]. This subcloned plasmid pCH-
After digestion of 15 with EcoRI, it was electrophoresed and developed on a 1.5% agarose gel. As a result, an inserted fragment (CH-15) of about 380 bp was confirmed (FIG. 1).

(6) Southern blot analysis using CH-15 Southern blot analysis using CH-15 was performed as follows. Chimpanzee normal and US NIH F strain infection N
Chromosomal DNA was purified from the liver of ANBH acute phase (8 weeks after NANBH virus inoculation) and from normal human leukocytes.
g was digested with EcoRI, electrophoresed and spread on a 2% agarose gel, and transferred to an NC filter. This filter was subjected to Southern hybridization using a CH-15 probe labeled with [ ³² P] by the multiprime method (FIG. 2).
As can be seen from this figure, the CH-15 probe, after autoradiography for one week,
Reacts with -15 clones, but normal and NANBH acute phase chimpanzee chromosomal DNA or normal human chromosome D
It did not react with NA. From this, it is considered that CH-15 is not a clone derived from human chromosomal DNA but is derived from an exogenous nucleic acid such as a virus.

(7) Nucleic acid base sequence and amino acid sequence of CH-15 (A) Determination of base sequence of CH-15 clone [α- ³² P] dCTP (800 Ci / mmol) was used for the reaction. Polymerase reaction with Klenow fragment was performed with Takara Shuzo 7DEAZA sequencing kit. 4 hours 18 using 8% polyacrylamide-8M urea gel
It electrophoresed at 00V 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. 3 and 4, respectively.

As a result of searching the obtained base sequence and amino acid sequence in a database (described above), there were no viruses, bacteria or other substances showing high homology. In addition, as a result of comparison with the nucleotide sequence and amino acid sequence of the non-A non-B hepatitis virus published by Chiron, a region having a homology of 83.1% with the nucleotide sequence of the CH-15 clone and 89.5% with the amino acid sequence was found by Chiron. It was present in the published sequence (see FIGS. 5 and 6).

(8) Reactivity with non-A non-B hepatitis patient serum in the antibody measurement method using 29mer and 37mer Based on the amino acid sequence encoded by CH-15, the following synthetic peptide consisting of 29 amino acids (29mer) and 37 amino acids A synthetic peptide (37mer) was synthesized.

A 430A peptide synthesizer manufactured by Applied Biosystems was used for the synthesis, and an HPLC column for purification of the synthetic peptide (Aquapore Prep-10, C-8, 300A pore siz) was used for the purification.
e, 20um spherical silica, 10mm ID × 250mm, Applied Biosystems) was used. When the composition of the purified synthetic peptide was analyzed according to a conventional method, it was confirmed that the desired peptide was synthesized.

Each synthetic peptide was dissolved in PBS (0.1 M PBS) to 1 μg / ml, and 200 μl was added to each well of the immunoplate.
Then, the mixture was reacted at 4 ° C. overnight, washed with PBS containing 0.01% Tween 80 (PBS-T), and coated with 0.1% BSA (bovine serum albumin) and a PBS solution at 250 μ / well. Thereafter, 200 μl of 0.1% BSA PBS-T was added to each well, 20 μl of a plasma sample was added thereto, and the mixture was slightly stirred with a plate mixer and reacted at 37 ° C. for 1 hour.

Next, after washing with PBS-T, an anti-human IgG HRP conjugate was added at 200 μ / well, and reacted at 37 ° C. for 1 hour.

After washing with PBS-T, 200 μl of TMBZ (3 ′, 3 ′, 5 ′, 5 ′,-tetramethylbenzidine hydrochloride) substrate solution was added to each well, and reacted at 37 ° C. for 30 minutes. 50μ /
The reaction was stopped by adding wells. This was measured with a microplate reader at two wavelengths of 450 nm / 650 nm.

As a result, when the above-mentioned immunosandwich assay was performed on 165 human plasma suspected to be non-A non-B hepatitis virus-infected blood, 122 samples out of 165 samples were positive in the measurement method using a 29-mer. In the measurement method using a 39-mer, 135 out of 165 samples were positive. That is, the non-A non-B hepatitis virus antibody assay using the 39-mer of the present invention is an assay with an extremely high detection rate.

Such a non-A non-B hepatitis virus antibody measurement method using the peptide of the present invention is an extremely excellent measurement method with less nonspecific reaction as compared with the conventional method.

[Brief description of the drawings]

FIG. 1 shows the ECH of pCH-15 cloned in the present invention.
FIG. 2 is a schematic diagram of a coRI inserted fragment after 1.5% agarose gel electrophoresis. FIG. 2 is a schematic diagram of Southern hybridization between CH-15 cloned in the present invention and chromosomal DNA of human and chimpanzee. FIG. 3 shows the nucleotide sequence of a nucleic acid fragment encoding a non-A non-B hepatitis virus antigen cloned in the present invention. FIG. 4 shows the amino acid sequence encoded by the non-A non-B hepatitis virus nucleic acid fragment cloned in the present invention. FIG. 5 shows a comparison between the nucleotide sequence of the non-A non-B hepatitis virus (HCV) gene published by Chiron and the nucleotide sequence of the CH-15 clone. The upper row shows the nucleotide sequence of the HCV genome, and the lower row shows the nucleotide sequence of CH-15 of the present invention. Common bases are indicated by *. FIG. 6 shows a comparison between the amino acid sequence encoded by the non-A non-B hepatitis virus (HCV) gene published by Chiron and the amino acid sequence of the CH-15 clone. The upper row is HC
Amino acid sequence encoded by V genome, lower row is C of the present invention
This is the amino acid sequence encoded by H-15, and a common amino acid is indicated by *.

──────────────────────────────────────────────────続 き Continued on the front page (51) Int.Cl. ⁷ Identification code FI C12R 1:92) Examiner Toshio Uchida (56) References WO 89/4669 (WO, A1) (58) Fields searched (Int. .Cl ^7, DB name) C12N 15/11 -. 15/62 C07K 14/00 - 14/825 GenBank / EMBL / DDBJ / G eneSeq

Claims (7)

(57) [Claims] 1. A nucleic acid fragment encoding a non-A non-B hepatitis virus antigen peptide comprising any one of the following amino acid sequences (a) to (c): 2. The nucleic acid fragment according to the above (1), comprising the following nucleotide sequence. 3. A nucleic acid fragment comprising the amino acid sequence of any one of the following (a) to (c) and encoding a peptide having a non-A non-B hepatitis virus-specific epitope. 4. The nucleic acid fragment according to the above (3), comprising the following nucleotide sequence. 5. A peptide obtained by chemical synthesis, which comprises an amino acid sequence of the following (a) or (b) and has a non-A non-B hepatitis virus-specific epitope. 6. A method for immunologically detecting a non-A non-B hepatitis virus-related antibody, comprising using the peptide according to the above (5). 7. A nucleic acid probe for detecting a non-A non-B hepatitis virus gene, comprising a nucleic acid fragment represented by the following nucleotide sequence or a sequence complementary to the nucleotide sequence: The nucleic acid probe.