JP2818761B2 - Nucleic acid fragment encoding non-A non-B hepatitis virus antigen and use thereof - Google Patents

Description

Description: FIELD OF THE INVENTION The present invention relates to a gene fragment encoding a viral antigen of a hepatitis virus that is not a type A or type B virus (non-A non-B hepatitis virus), The present invention relates to hepatitis B virus antigen peptides and methods for using them.

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 diagnostic 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., Lance.
tI 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 p818p824, (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 experiments, Tabot, 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 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, ie, had a similar floating density of the 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. Suspicious, the infection model is chimpanzee,
There are only marmosets.

Recently, there was a report that Chiron in the United States captured non-A, non-B hepatitis virus cDNA [Choo, Q
et al., Science, 244, P359-362 (1989), Kuo, G. et a
l., Science, 244, p362-364 (1989)], the properties of the virus itself, sequence information, and the properties of the virus-constituting proteins have not yet been elucidated.

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 hepatitis B virus antigen peptide sequence was successfully cloned.

That is, the present inventors have finally conducted a non-A non-B hepatitis treatment by using a high GPT plasma of a blood donor and conducting an immunoscreening method incorporating a new molecular genetic technique different from the conventional immunoserologic method. The gene encoding a peptide unique to the virus was cloned. Furthermore, it has been confirmed that the expression product obtained by expressing this gene fragment using a gene recombination technique specifically reacts with the serum of a non-A non-B hepatitis patient even at the protein level, thereby completing the present invention. Reached.

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 carriers of non-A non-B hepatitis has been confirmed,
In transfusion, the higher the donor's GPT value, the higher the incidence of post-transfusion non-A, non-B hepatitis. Therefore, it is estimated that plasma with a high GPT value has a high carrier frequency. Therefore, we pooled relatively high-available GPT-high plasma from Japanese blood donors for study. In addition, non-A Japanese
The plasma of chimpanzees that have developed non-A non-B hepatitis by inoculating the serum of a non-B hepatitis patient can also 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, plated 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,
Remove 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 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 a 0.28 Kbp insert (C825) was confirmed by agarose gel electrophoresis.

Liver of normal life and non-A, non-B hepatitis acute chimpanzee, as well as chromosomal DNA was purified from normal human leukocytes, after agarose electrophoresis, and subjected to Southern hybridization using the ³² P-labeled C825 clones Was.
C825 did not react with any of the DNA, thus indicating that C825 was not chromosomal DNA.

Also, Japanese GOT, GPT high plasma pool (non-A non-B
Hepatitis B infectivity has been confirmed in chimpanzee infection experiments), RNA was extracted from chimpanzee plasma passaged from non-A non-B hepatitis of the NIH strain derived from the United States of America and normal human plasma in Japan, and cDNA was synthesized. PCR using part of the nucleotide sequence of the C825 and C825 clones as primers [SAiki et al. Science 23
9, p487- (1987)]. Similarly from normal human liver
DNA was extracted and a PCR reaction was performed using the same primers.
As a result, only the pool of GOT and GPT high plasma in Japanese
825 base sequences were detected. This suggests that the non-A non-B hepatitis virus containing the nucleotide sequence of C825 that we captured has a significant difference in nucleic acid sequence from the non-A non-B hepatitis virus of the US NIH-derived F strain. .

The DNA sequence of the C825 clone of the present invention was determined by the dideoxy method. As a result, the C825 clone was a total 269 bp cDNA fragment derived from the non-A non-B hepatitis virus gene, and its nucleotide sequence was as shown in FIG. This DNA
When the sequence was read back to amino acids, only one frame did not have a stop codon in the middle, indicating that this was part of the open reading frame. When this base sequence and amino acid sequence were searched in a database (Genetyx-CD software development 1989), there were no known viruses, bacteria, or other homologues.

From this amino acid sequence, the hydrophilicity / hydrophobicity pattern of the peptide encoded by C825 was analyzed based on the method of HOPP & WOOD et al. As a result, the results shown in FIG. 5 were obtained, and it was confirmed that this peptide region was a peptide having strong hydrophilicity as a whole. Furthermore, among the peptides consisting of 89 amino acids, 4
It was confirmed that two regions existed.

As described above, the cDNA fragment obtained by the present invention is a non-A non-B
The fact that it encodes a peptide region having strong hydrophilicity among hepatitis hepatitis virus antigens was considered to be very significant from an immunological point of view. In addition, since such a hydrophilic peptide is easy to handle, it is very useful from the viewpoint of practicality.

In the present invention, as a preferred example of the non-A non-B hepatitis virus antigen peptide, not only a peptide consisting of 89 amino acids as shown in FIG. 4 is disclosed, but also a non-A non-B hepatitis virus antigen in particular. Also disclosed are the four strongly hydrophilic non-A non-B hepatitis virus antigen epitopes described above, which are believed to be closely related to gender.
Such an antigenic epitope is the amino acid sequence of (A) to (D) below.

As shown in the analysis pattern of FIG. 5, it is confirmed that the peptide region of the above amino acid sequence is a peptide having particularly strong hydrophilicity.

To further confirm the association with non-A non-B hepatitis,
The plaque immunoassay and the dot immunoassay for C825 were performed using the sera of many hepatitis patients and normal persons. As a result, antibody-positive individuals were detected in non-A non-B hepatitis patients at a higher rate than in normal, hepatitis B, and other hepatitis groups. Proven.

The gene sequence of the present invention can be expressed by using an appropriate expression system and used for an antibody test of non-A non-B hepatitis virus. It can also be used to detect non-A non-B hepatitis virus in liver tissue of non-A non-B hepatitis infected patients.

Furthermore, the non-A, non-B hepatitis virus obtained in the present invention is 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 nucleic acid fragments encoding the non-A non-B hepatitis virus antigen peptide of the present invention, the non-A non-B hepatitis virus antigen peptide, and various methods for detecting the non-A non-B hepatitis virus using these, 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
0 or more human pooled plasma (approximately 8.2)
Concentrated by a factor of two. First, human pool plasma was subjected to rough centrifugation to remove insolubles. 1/10 volume of 5M sodium chloride solution, and then 1/10 volume of 40% (W / W) polyethylene glycol solution (PEG6000, manufactured by Wako Pure Chemical Industries, average molecular weight 75000) with stirring at 4 ° C. Was added. After standing for 1 hour, the supernatant was removed by centrifugation at 7000 rpm for 20 minutes, and a TNE solution (10 mM Tris-HCl, pH 7.4, 1 mM EDTA,
(140 mM NaCl) was added and redissolved. Add this solution to sucrose
20%, 15%, 10% and 5% TNE solutions were layered on top of the stepwise layered centrifuge tubes and ultracentrifuged at 80000 × G at 4 ° C. for 12 hours. After separation, the supernatant was removed and the sediment was washed with 8 ml of PBS.
To give a 1000-fold concentrate of GOT and GPT high-value human pooled 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% sarkoxy) was added, and the mixture was stirred and extracted with phenol / chloroform, and the total 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 this 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 glycogen present in this RNA and impurities considered to be present in a trace amount, QI
Purification scanning was performed using AGEN pack-100 (manufactured by DIAGEN).

(3) Construction of cDNA Library All 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 1.2 × 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 used for screening the cDNA library 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 2LB medium, and further cultured at 37 ° C overnight. Transfer the culture to a centrifuge tube, 9000 rpm, 10 minutes, 4
After centrifugation at ℃, the supernatant was removed to obtain a precipitate. This sediment
4 ml of RIPA solution per 1 g (1% sodium deoxycholate, 1% Triton X-100, 0.3 M NaCl, 0.1% SDS, 0.1 MT
ris-HCl (pH 7.5, 1 mM PMSF) was added to solubilize it, and the mixture was further centrifuged at 9000 rpm for 10 minutes at 4 ° C., and the supernatant was used as E. coli lysate.

(B). Preparation of Replica Filter for Antibody Screening From cDNA library constructed from RNA in GOT, GPT high 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 10,000PFU of phage per E. coli Y1
Infect 090 at 37 ° C for 15 minutes, Top Agar 40ml (0.7% Ag
ar, 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. 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 filters 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 The lysate was added to the E. coli, and the non-specific reaction was absorbed overnight at 4 ° C., and further diluted 2-fold with PBS. ], And reacted while shaking at room temperature. Two hours later, PBS-T (0.05% T
After washing the replica filter three times with a PBS solution containing ween20 for a total of 15 minutes each time, an incubation buffer containing a 1000-fold diluted peroxidase-labeled anti-human IgG and an IgM goat antibody (MBL, Fab). (1
% Bovine serum albumin) and reacted at 37 ° C. with shaking. After one hour, the plate was washed with PBS-T for 115 minutes each time, four times in total, and then washed with PBS for 5 minutes, and then the color developing solution [0.02% DAB (Sigma), 0.1% NiCl ₂ .6H ₂ O, 0.0%
5% H ₂ O ₂ ]. Phages corresponding to plaques developed on the NC filter were selected and subjected to secondary screening. That is, 200PFU of each phage selected in the primary screening was separately
At the same time, the cells were infected with Escherichia coli Y1090 and re-rolled on a 90 mm petri dish (manufactured by Becton Dickinson) LB plate to prepare a replica filter. These were subjected to antibody screening by the above-described method to obtain three clones (C8-2, C10-1, C16-1) of phage which react with the chimpanzee plasma in the recovery phase and the carrier phase of the NANBH with good reproducibility.

(5) of each clone using antibodies in chimpanzee serum
Examination of specificity for NANBH For the three clones obtained in (4), (4).
A replica filter was prepared in the same manner as in the next screening, and a plaque assay was performed using the NANBH recovery phase, carrier phase and normal chimpanzee serum, or IgG fraction purified by ammonium sulfate precipitation and then DEAE-cellulofin column (manufactured by Seikagaku Corporation). Was done. 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,
Escherichia coli lysate was added in an amount of 0, and used at 4 ° C. overnight after absorbing nonspecific reaction.

As a result of the plaque assay, two clones (C10-1, C16
Regarding -1), it also reacted with antibodies in normal chimpanzee serum, and the specificity for NANBH was low. However, C8−
2 is chimpanzee serum or IgG from NANBH carrier
It reacted with the fraction at a high rate, and did not react at all with serum or IgG fraction of normal chimpanzee. Table 1 shows the results.

From these results, it can be said that C8-2 is a clone highly specific to chimpanzee serum in the NANBH carrier phase.

The C8-2 phage DNA was purified [see Experimental Medicine Extra Edition, Genetic Engineering Summary 5 (11), pp. 31-32 (1987)], digested with the restriction enzyme EcoR I (manufactured by Toyobo), and cut into the pUC118 vector EcoR I. Site and subcloned [Douglas Hanahan, J. Mol. Biol. 166 , P557-580 (198
3)]. This subcloned plasmid pC825
Was digested with EcoRI, and electrophoresed on a 2% agarose gel. As a result, an inserted fragment (C825) of about 0.28 Kbp was confirmed (FIG. 1).

(6) Southern blot analysis using C825 Southern blot analysis using C825 was performed as follows. Chimpanzee normal and US NIH F strain-infected NAN
Chromosomal DNA was purified from liver in the acute phase of BH (8 weeks after inoculation with NANBH virus) and leukocytes from normal humans, and 20 µg each.
Was cut with EcoR I, spread on a 1% agarose gel by electrophoresis, and transferred to an NC filter. The filter was subjected to Southern hybridization using a C825 probe labeled with [ ³² P] by the multiprime method (FIG. 2). As can be seen from this figure, the C825 probe reacts with the C8-2 clone before subcloning when autoradiographed for one week, but does not react with the chromosomal DNA of normal and NANBH acute-phase chimpanzees or normal human chromosomal DNA. Did not react. From this, C825 is the human chromosome DN
It is considered that the clone is not derived from A but derived from an exogenous nucleic acid such as a virus.

(7) Nucleic acid base sequence and amino acid sequence of C825 (A) Determination of base sequence of C825 clone [α- ³² P] dCTP (800 Cim mol) was used for the reaction with plasmid DNA incorporating the C825 gene fragment as a template. . 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 nucleotide sequence and predicted amino acid sequence The nucleotide sequence obtained as a result of the above and the result of decoding the amino acid sequence predicted therefrom are shown in FIGS. 3 and 4, respectively.

The hydrophilicity / hydrophobicity profile of the predicted amino acid sequence of C825 is shown in FIG.

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.

(8) C8 using Polymerase Chian Reaction (PCR)
Detection of 25 nucleotide sequence GOT, high GPT human pooled plasma used as material for taking C825 clone, chimpanzee plasma inoculated with US NIH-derived NANBH strain F (confirmed infectivity), normal human plasma And chromosomal DNA from human liver
The C825 base sequence was detected using the CR reaction. First, 1 ml of each plasma was precipitated using a 5 M sodium chloride solution and a 40% (w / w) polyethylene glycol solution in the same manner as in (1), and a 500 μg guanidinium thiocyanate solution was added to the precipitate. Was added, and the total nucleic acid was purified by phenol / chloroform extraction and ethanol precipitation. This was added to 50 mM Tris-HCl pH 8.3, 6 mM MgCl ₂ , 40 mM KCl, 1 mM
DTT, 1mM dNTPs, 1.3KU / ml, RNasin, 30mg / ml random primer, 4KU / ml reverse transcription oxygen (BRL) 20μ mixture
The reaction was carried out at 37 ° C. for 1 hour and 30 minutes. This reaction solution 1
Take μ and 10 mM Tris-HCl pH 8.3, 50 mM KCl, 1.5 mM Mg
Cl ₂ , 0.01% (w / v) gelatin, 100 nM dNTPs, 250 nM primer (position shown in FIG. 6) 20 U / ml Taq polymer
In 50 μl of ase mixture, 94 ° C; 30 seconds, 55 ° C; 30 seconds, 72 ° C; 1
The reaction was carried out for 40 cycles with one minute as a cycle (using a thermal cycler manufactured by Perkin-Elmer Cetus). For chromosomal DNA derived from human liver, 10
μg in a reaction solution of the above composition under the same conditions as above.
The reaction was performed. After the PCR reaction, 5 μ of each sample was taken, developed on a 2% agarose gel by electrophoresis, and transferred to an NC filter. An oligo probe in C825 labeled with [ ³² P] of this filter (the position is shown in FIG. 6)
Was used for hybridization. As a result C8
The 25-base sequence was detected in GOT and GPT high-value human pooled plasma, but was not detected in normal human plasma, chimpanzee plasma of F strain derived from NIH in the United States, and chromosomal DNA (FIG. 7). .

(9) Examination of the specificity of the C825 clone for non-A non-B hepatitis (A) Plaque immunoassay In the same manner as in (4), using the serum of a Japanese hepatitis patient,
The specificity of 25 clones for non-A, non-B hepatitis was examined by plaque assay. The results are shown in Table 2 below.

(B) Dot immunoassay E. coli Y1089 containing ampicillin (Ap) (50 μg / ml) L
After culturing in B, the λgt11 phage and C8-2 phage are adsorbed at a multiplicity of infection (moi) of 10 at 37 ° C. for 15 minutes. A colony is formed on an LB plate (containing Ag), and the colony is picked up with a toothpick, transferred to an LB plate at 30 ° C. and 42 ° C., and cultured. Colonies that grew at 30 ° C but did not grow at 42 ° C were selected. lysogen derived from λgt11 and λC8-2
λ11 and LλC8-2.

These lysogens are placed in 10 ml LB medium (containing Ag) at 30 ° C.
After lactose operon expression is induced by adding 1 mM IPTG, the cells are collected. 1 ml RIPA
The solution was dissolved in the solution, 1 g of glass beads were added, and the mixture was crushed with a vortex mixer. The supernatant was collected and used as a lysogen extract in a dot assay.

5 μl of the prepared lysogen extract was spotted on a nitrocellulose filter. After drying, the steps from the blocking reaction to the coloring reaction were performed in the same manner as in (4). as a result,
Sera that reacted with the LλC8-2 extract but did not react with the Lλ11 extract were determined to be positive (see FIG. 8). Table 3 shows the results of this determination.

As described above, in the non-A non-B hepatitis patient group, the positive rate was as high as 51.7%, whereas the normal person, B
Positive rate 0.0%, 11.0 for patients with hepatitis B and other hepatitis
% And 0.0%, the C825 clone of the present invention is non-A
It was shown to be non-hepatitis B specific.

[Brief description of the drawings]

FIG. 1 shows the Eco-DNA of pc825 cloned in the present invention.
FIG. 3 is a schematic diagram of a RI insertion fragment after 2% agarose electrophoresis development. FIG. 2 is a schematic diagram of Southern hybridization between C825 cloned in the present invention and human and chimpanzee chromosomal DNA. FIG. 3 shows the nucleotide sequence of C825 cloned in the present invention. FIG. 4 shows the entire amino acid sequence encoded by C825 cloned in the present invention. FIG. 5 shows the hydrophilicity / hydrophobicity profile of the peptide encoded by C825 based on the amino acid sequence. FIG. 6 shows C82 used in the PCR reaction in Example (8).
It shows the positions of primers and oligo probes in the 5-base sequence. FIG. 7 is a schematic diagram of hybridization of a gene amplified from human chromosomal DNA and nucleic acid in serum using PCR in a primer of the base sequence of C825 cloned in the present invention and using a primer.

──────────────────────────────────────────────────続 き Continued on the front page (51) Int.Cl. ⁶ Identification code FI C12Q 1/68 C12Q 1/68 A G01N 33/576 G01N 33/576 Z // (C12P 21/02 C12R 1:19) (56 References WO 89/4669 (WO, A) WO 91/1376 (WO, A) SCIENCE, Vol. 244, (21 April 1989); 359-362 (58) Fields investigated (Int.Cl. ⁶ , DB name) C12N 15/00-15/90 C07K 14/18 C07K 16/10 C12P 21/00-21/08 C12Q 1/68 G01N 33 / 576 BIOSIS (DIALOG) GenBank / EMBL / DDBJ (GENETYX) WPI (DIALOG)

Claims (6)

(57) [Claims] An amino acid sequence comprising the following amino acid sequence, or an amino acid sequence in which one or several amino acids have been deleted, substituted or added, and having a non-A non-B hepatitis virus-specific epitope. Non-A
A nucleic acid fragment encoding a non-hepatitis B virus antigen peptide. 2. The nucleic acid fragment according to the above (1), which comprises the following nucleotide sequence or a nucleotide sequence in which one or several nucleic acids have been deleted, substituted or added in said nucleotide sequence. 3. An amino acid sequence comprising the following amino acid sequence, or an amino acid sequence in which one or several amino acids have been deleted, substituted or added, and has a non-A non-B hepatitis virus-specific epitope. Non-A
Non-hepatitis B virus antigen peptide. 4. The peptide, wherein the nucleic acid fragment according to the above (1) or (2) is incorporated into an appropriate expression vector,
The non-A, non-B hepatitis virus antigen peptide according to the above (3), which is a peptide obtained by expressing this in a host cell. 5. An anti-non-A, non-B hepatitis virus antibody prepared using the peptide of (3) or (4) as an antigen. 6. A method for immunologically detecting non-A, non-B hepatitis virus, comprising using the antibody according to the above (5).