JPH0466596A - Non a, non b hepatitis virus antigen peptide, nucleic acid fragment coding same and utilization thereof - Google Patents

Abstract

NEW MATERIAL:Non A, Non B hepatitis virus antigen peptides containing an amino acid sequence of the formula or a part thereof. USE:A diagnostics or a remedy for non A, non B hepatitis. PREPARATION:For example, plasma of a patient of non A, non B hepatitis is concentrated and the whole nucleic acids in the plasma are separated using the guanidinium thiocyanate method. The separated nucleic acids are treated with DNase to decompose human-derived DNA and RNA is separated. cDNA is synthesized using the separated RNA as a template and cDNA library is formed thereby. The resultant cDNA library is subsequently screened using a prove to select a positive clone containing cDNA capable of coding the non A, non B hepatitis virus antigen peptide and DNA is extracted therefrom. The extracted DNA is treated with a restriction enzyme, then combined with a manifestation vector and introduced into a host for transformation thereof. The obtained transformant is cultured, thus obtaining the objective antigen peptide of the formula.

Description

[Detailed Description of the Invention] tJLL, March 11, 2013 The present invention relates to antigen peptides of viruses that cause hepatitis of serotypes that are neither type A nor type B (non-A, non-B hepatitis virus), gene fragments encoding the same, and Regarding usage. ' Liao Ju It has long been known that there are two types of viral hepatitis: hepatitis A (infectious hepatitis) and hepatitis B (serum hepatitis). This is mainly based on differences in infection routes;
It has been confirmed that hepatitis A causes epidemics through oral infection, and hepatitis B is primarily transmitted through blood. These two hepatitis-causing viruses have already been isolated and identified, and hepatitis A virus is an RNA virus with a diameter of 27n- that belongs to the picornavirus [Finest
on, S, between, et al,, 5science 1
82 p1026 (1973)], on the other hand, the hepatitis B virus was found to be a DNA virus with a diameter of 42 nm and an envelope belonging to the hepadnavirus. [Daoe, 0. s, et al.

Lancet, I p695 <1970)] and
At present, immunoserological diagnostic methods for these hepatitis viruses have been established.

As definitive diagnostic methods for these two hepatitis viruses have been established, the existence of non-A, non-B hepatitis that does not belong to either of them has become clear [Prlnce, A.

Lancet, et al, Lancet, I p241
(1974>].

Post-transfusion hepatitis is caused by hepatitis B virus surface antigen (HBsAg).
) has been significantly reduced with the introduction of screening methods, but has not been reduced to zero.
No evidence of hepatitis B or hepatitis B infection was found. For this reason, this hepatitis is generally called non-AIF, hepatitis B.

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

Separately, there is a second viral non-A, non-B virus that is prevalent through oral infection in India, Burma, Afghanistan, and Africa. ! ! It became clear that he had hepatitis [Khuroo, M, S, Am, J, M
ed.

68 p818-824. (1980)], which is commonly referred to as waterborne, or epidemic non-A, non-B hepatitis. Although this hepatitis epidemic has not been observed in Japan, there seems to be some cases of hepatitis being imported from endemic areas by travelers [Fukuhara et al., Abstracts of the 25th Japan Hepatology Society Conference, p. 151 (1989 ) 1.

The present invention relates to the former serotype non-A, non-B hepatitis virus that is mainly transmitted through blood,
This virus is referred to herein as a non-A, non-B hepatitis virus.

The virus itself for this non-A, non-B hepatitis has not been isolated and identified, and therefore no diagnostic, therapeutic, or preventive methods for this hepatitis have been established.

Furthermore, the only way to diagnose this hepatitis was to make a diagnosis of exclusion.

That is, the patient's serum is tested for hepatitis A and B, for which diagnostic methods have been established, to deny these hepatitis cases, and to show hepatitis symptoms as part of systemic infection. Herbe entered Cytomegalo, denying the possibility of Epstein-Barr virus infection, drug-induced hepatitis, alcoholic hepatitis, and autoimmune hepatitis, and was diagnosed with non-All: B hepatitis.

It is known that the virus that causes hepatitis is infectious.
This was proven by an American research group in 1988 through infection experiments using chimpanzees [Tabor, E.

et al., Lancet, I p463 (197
8)], Despite many efforts around the world, the causative virus is still unknown even after more than 10 years. Almost every approach used in hepatitis A and B research has been used to search for viruses and associated antigen-antibody systems, including precipitation reactions, immunoelectrocurrent techniques, radioimmunoassays, immunofluorescence, and electron microscopy. However, I have not been able to obtain anything that is said to be very reliable.

The history of research into non-A, non-B hepatitis viruses can be said to be a history of expectations and disappointments. Many candidates for viruses or antigen-antibody systems have emerged, but they have been rejected one after another [Prince, A. M.,
^nn, Rev, 14icrobio1. , 37
．． p217. (1983) Ko.

A recent example is the retrovirus theory by 5eto et al. [
5eto, B, et tl,: Lancet, I
p941-943 (1984)], according to them,
Reverse transcriptase activity was detected in serum and blood products that have been proven to cause non-A, non-B hepatitis in chimpanzees, and sucrose density gradient centrifugation showed that this enzyme was found at 1.14 g/L. In other words, it had a floating density similar to that of retroviruses.Ce et al. reported that retrovirus-like particles were observed when patient serum was inoculated into chimpanzee liver primary culture cannons [Pr1
nce, AJ, et al. Lancet, I:
p1071-1075 (1984)], but reverse transcriptase activity was denied by follow-up tests by Ollinger et al.
, Lancet.

Furthermore, the virus particles observed by Prince et al. were ruled out as myxovirus contamination.

Problems that make research on non-A, non-B hepatitis difficult include the low virus concentration in the serum of 102 to 103, and the fact that some chimpanzees have been reinfected with the same inoculum.
The existence of antibodies is questionable, and the only experimental models for infection are chimpanzees and marmosets.

Recently, there was a report that the US company Chiron had captured the cDNA of a non-A, non-B hepatitis virus.
o, Q et al, , 5science,
244. P2S5-362 (1989), Kuo
, G. et al., 5science, 244.
p362-364 (1989) 1, although part of its base sequence has been published (European Patent EP 31821 (y
The properties of AE, the virus itself, and the constituent proteins of the virus have not yet been clarified.

Generally, new methods will be completely different due to differences in viruses due to differences in their immunoserological properties and molecular genetic properties. In addition, differences in strains have some differences in immune serological properties, so using the same diagnostic method will result in differences in detection sensitivity due to differences between strains, and in vaccines, differences in immunogenicity and ability to protect against infection.8 Molecular genetics It is generally known that in conventional diagnostic methods, such as DNA probe diagnosis, hybridization between a probe and a viral nucleic acid is not practical unless the homology at the nucleic acid level is extremely high. In other words, due to differences at the nucleic acid level between strains,
There may be cases in which DNA hybridization does not occur and DNA probe diagnosis cannot be performed effectively.

Hepatitis B, which is a well-known and well-analysed serotype of hepatitis, has major hepatitis virus subtypes in each region, such as Europe, America, and Southeast Asia. Type> is known to exist, therefore, non-A type
Even among non-B hepatitis viruses, there may be regionally specific virus species or strains.

Therefore, in order to establish diagnostic and preventive methods for non-A, non-B hepatitis viruses that are prevalent in specific regions, especially Japan, it is necessary to identify the major non-A, non-B hepatitis virus strains in Japan. .

Under these circumstances, the present inventors conducted repeated research aimed at cloning the causative virus of non-A, non-B hepatitis or its viral genes. We cloned a gene encoding the antigenic peptide sequence of hepatitis B virus and confirmed that the peptide encoded by this gene reacts specifically with non-A, non-B hepatitis virus.

That is, the present inventors obtained non-A11:B by an immunoscreening method that incorporates a new molecular genetic method different from the conventional immunoserological method using GPT-high plasma from blood donors. The present invention provides a peptide unique to the hepatitis virus, a gene fragment encoding the same, and methods for using these.

]" When cloning nucleic acid fragments as the object of the present invention, livers of Japanese people infected with non-A It" B hepatitis and livers of chimpanzees infected with non-A non-B hepatitis were used as research materials. Extract mRNA and cD using
It is conceivable to synthesize NA and select virus-specific cDNA from it by subtraction with chromosomal DNA. However, it is extremely difficult to secure sufficient quantities of good experimental materials necessary for this purpose.

Another possible research material would be plasma from non-A, non-B hepatitis infected individuals or infected chimpanzees.

The existence of non-A, non-B hepatitis carriers has been confirmed in humans, and the higher the donor's GPT value during blood transfusion, the higher the incidence of non-A, non-B hepatitis after blood transfusion. It is estimated that the frequency of carriers is high.

Therefore, we pooled high GPT plasma from Japanese blood donors, which is available in relatively large quantities, and used it as research material. In addition, serum from one Japanese patient with non-A, non-B hepatitis was inoculated and IF
Plasma from chimpanzees that have developed A, non-B hepatitis can also be used, but some problems remain due to the current availability of chimpanzees.

As mentioned above, the concentration of non-A, non-B hepatitis virus in plasma is estimated to be only about 102 to 10 2 O), so extraction of viral nucleic acid and synthesis of cDNA requires 10
It is necessary to concentrate the virus approximately 00 times. However, human plasma is a protein solution of around 7%, and it is impossible to simply concentrate it, and it is necessary to concentrate the virus while removing protein. Precipitate formation using a precipitant such as polyethylene glycol (PEG), which we used, is a mild method that can be performed relatively easily, is suitable for processing large amounts of plasma, and causes less virus deactivation.

Other methods that can be used include veretting using ultracentrifugation, salting-out ultrafiltration by adding salts such as ammonium sulfate, and gel chromatography.

The plasma thus concentrated approximately 1000 times is treated with guanidinium thiocyanate-1-7', extracted with phenol/chloroform, and then subjected to ethanol precipitation to purify all the nucleic acids in the concentrated plasma. Next, 1) Contaminant human-derived DNA is degraded using NA degrading enzyme, and RNA is purified by phenol/chloroform extraction and ethanol precipitation.

cDNA is synthesized from the purified RNA and inserted into a λgtll vector to create a cDNA library.

Escherichia coli is infected with the λ phage, plated on bacterial culture plates, and cultured at 42°C for several hours. Thereafter, cover with a 21-hole cellulose filter (NC filter) and culture for several hours, then remove the NC filter and take 2 replicas.

This replica is treated with a blocking solution, pre-cleaned with PBS, etc., and then subjected to immunoscreening.

That is, the replica was reacted with non-A, non-B hepatitis convalescent or acute phase human or chimpanzee serum, and PBS
After washing, react with enzyme-labeled anti-human IgG or IgM, and after washing, react with a substrate solution to develop color. Select phages corresponding to colored plaques and perform secondary screening to obtain reproducible clones. Ta.

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

As a result of performing a plaque immunoassay using IgG from chimpanzees in the convalescent stage of non-A, non-B hepatitis, carrier stage, and normal stage, we were able to obtain clones with high specificity for the non-A, non-B hepatitis carrier stage. This clone was subcloned and approximately 380 bp was analyzed by agarose gel electrophoresis.
The inserted fragment (CO-15) was confirmed.

normal and non-A, non-B hepatitis acute stage liver of chimpanzees;
In addition, chromosomal DNA was purified from leukocytes of normal people, and after performing agarose electrophoresis, $2p-labeled CH-15
Southern hybridization was performed using the clone. CH-15 did not react with any DNA, and it was therefore determined that CI+-15 was not chromosome-derived DNA.

The DNA sequence of the present invention (7) CH-15 clone was determined by the dideoxy method. As a result, a total of 372 C1,-15 clones were derived from non-A, non-B hepatitis virus genes.
It was a cDN^ fragment of bp, and its base sequence was as shown in Figure 3. This nucleotide sequence and the amino acid sequence deduced from it are stored in a database (GenetyxC).
D Software Development 1989), there were no viruses, bacteria, or anything else known to date that showed homology. Furthermore, as a result of comparing the nucleotide sequence and amino acid sequence of non-A, non-B hepatitis virus (Hcv) published by Chiron Corporation of the United States, the nucleotide sequence is 83.1% similar to that of CH-15, and 89.5% is similar to the amino acid sequence. A region with homology was found in the sequence published by Chiron. From this result, it was presumed that this clone was the same type as the one cloned by Chiron, and was a non-A, non-B hepatitis virus.

Furthermore, from among the amino acid sequences encoded by Cto15,
When the following two peptides (29■er and 37ser) with different lengths were synthesized and reacted with non-A, non-B hepatitis patient serum, they reacted very specifically with non-A, non-B hepatitis patient serum. This was confirmed.

29 ■er: Glu Phe Asp Glu Met Glu G
lu Cys^la Ser olds Leu Pro
Tyr Vat Glu Gln Gly
! 4et GinLeu Ala Glu
Gin Phe Lys Gin Lys
Ala7ser Pro Asp Arg Glu Val Leu T
yr Arg Glu Phe^sp Glu Met
Glu Glu Cys Ala Ser [(is
LeuPro Tyr Val Glu Gln G
ly Met Gln Leu AlaGlu Gln
Phe Lys Gin Lys^1& That is, the present invention discloses an antigenic epitope with particularly high specificity among the amino acid sequences encoded by CH-15.

Among the above peptides, especially non-A, non-B using 37■er
In the hepatitis virus antibody measurement, even when compared with a similar measurement method using 29■er, extremely superior results were shown in terms of both specificity and sensitivity.

The gene sequence of the present invention can be expressed using a known appropriate 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, either a prokaryotic or eukaryotic expression system can be used, and if necessary, a fusion protein with an appropriate peptide can be made. On the other hand, chemical synthesis methods are convenient for preparing relatively short peptides consisting of specific epitopes. Such a peptide of the present invention can be used for antibody testing for non-A, non-B hepatitis viruses, and can also be used by immunizing animals with the peptide to produce antibodies specific to the peptide. Non-A11l
It is also possible to detect non-A, non-B hepatitis viruses in the liver tissue of patients infected with hepatitis.

Furthermore, the non-A, non-B hepatitis virus obtained in the present invention is
It is considered to be extremely useful for producing vaccines for preventing infection.

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

The non-A, non-B hepatitis virus antigen peptide of the present invention, the nucleic acid fragment encoding the same, and various detection methods for non-A, non-B hepatitis viruses using these are particularly useful for non-A, non-B hepatitis in Japan. It is considered to be extremely useful in detecting viruses.

Hereinafter, the present invention will be explained in more detail with reference to Examples.

GP donated by the Japanese Red Cross Society and negative for EtBs antigen.
Human boule plasma (8.5ff) with a T value of 100 or more was concentrated 1000 times using the following method, and human 7-le plasma was roughly centrifuged to remove insoluble matter.
M sodium chloride solution, then 1/10 volume f) 40%
(W/11) Polyethylene glycol liquid (PEG60
011, manufactured by Wako Pure Chemical Industries, Ltd., average molecular weight 7500) was added at 4°C with stirring. -700 after 1 time jump
Centrifuge at 0 rpm for 20 minutes, remove the supernatant, and add TNE solution (10 d Tris-[
(C1, pH 7.4, bwM EDTA, 140mN
acI) was added and redissolved. Apply this solution to 20% of the adhesions.
%, 15%, 10% and 5% THE solutions were layered on top of a centrifuge tube in stages, and at 4°C and 80,000 x.
After 12 hours of ultracentrifugation, the supernatant was removed, and the precipitate was dissolved in 8-1 PBS to obtain a 1000-fold concentrate of Gffr-GPT-high human boule plasma.

First, 5 times the amount of guanidium thiocyanate solution (4M guanidium thiocyanate, 50mM Tris-HCI pH 7,6,
After stirring, add phenol/
Total nucleic acids in concentrated plasma were purified by chloroform extraction and ethanol precipitation using glycogen as a carrier.
Next, in order to degrade the human-derived DNA present in this total nucleic acid, RNase-free DNase 1.1 was added in the presence of 2sM vanadyl ribonucleotide complex.
5KU/ml (Boehringer/Mannheim), 50
-M TrisHCI pH7,4,1mM EDTA
, IOIIM MgCh mixture 400u! in j, 3
It was treated at 7°C for 30 minutes. Then 250mM EDT
The reaction was stopped by adding 16 μ of solution A and 8 Δ of 10% SDS, and the 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 a trace amount of impurities, Q! AGBN pack-100 (D
The purification operation was performed using the following product (manufactured by rAGEN).

cDNA--All of the RNA purified by the above method was converted into cDNA using cDNA synthesis system plus (manufactured by Amajam).
8 After synthesis, the synthesized cDNA was cloned into λgtxt using cDNA cloning λgtll (manufactured by Amajam).
Cloned into a vector. As a result of in vitro packaging, a library of 1.8 x 10 @ plaque forming units (PFU) was obtained.

(4) Screening of NANBH virus clones in non-A, non-B hepatitis (NANBFI) convalescent and carrier stages The primary antibody used for screening the NANBH convalescent and carrier stages was chimpanzee plasma. Therefore, a high nonspecific reaction was expected. Therefore, in order to suppress this non-specific reaction, E. coli Y1090, which is used for the absorption operation of chimpanzee plasma for screening,
A lysate was prepared. That is, from a single colony, LB medium containing 50 m/sl of ampicillin [1% Bacto
-trytone (manufactured by Difco), 05% Bacto
-yeast extract (manufactured by Difco), 1%
Escherichia coli Y1090 culture 2011, cultured overnight at 37°C in 7,5'l of NaCl, pl, was added to 2Q LB medium and further cultured overnight at 37°C. This culture was transferred to a centrifuge tube and incubated at 9000 rpm. , centrifuged for 10 minutes at 4°C,
The supernatant was removed to obtain a precipitate. 41 R per gram of this sediment
IPA solution (1% sodium deoxycholate, 1% Tr
lton X-100, 0,3M NaC1,0,1%S
DS, 0. IM Tris-HCI pH7,5
.

B 1-2' 1
- ■G(rr, RNA in GPT-high human boulevard plasma concentrate)
From the cDN^ library constructed from
acto-tryptone, 0.5% Bacto
-yeast extract, 1% NaCl pl(
Take 100 OOPFU of phage per bacterial culture plate (manufactured by Nunc, 23C x 23cm) containing ampicillin for 7, 5, 50 rounds/meal, and use Escherichia coli Y1090.
Top Agar 4 was infected for 15 minutes at 37°C.
0 m1 (0,7% Agar, 1% Bac t.

tryptooe, 0.5% Bacto-yeas
extract, 1% NaCl, pH 7.5,
50Itg/ml ampicillin), and intercultured at 42°C for 4 to 5 hours. After that, a nitrocellulose filter impregnated with 10■-IPTG (manufactured by Sigma) (
NC filter: Manufactured by S&S, Code BA85
．． 23 cm x 23 cm), and culture was continued at 37°C.

After 3 hours, remove the NC filter from the plate and add PBS.
Wash with Blocking solution (5% skim milk, 0.5%).
Soak the replica filter in PBS solution containing 05% NJLN3 and shake overnight at 4°C.
After washing with BS, diluted 10 times with PBS.
B H chimpanzee pool plasma in convalescent and carrier phases (plasma for screening) [NANBH convalescent and carrier phase chimpanzee pool plasma in PBS
Dilute 1:20, add 1/20 volume of E. coli lysate, and add to 4°C.
Absorption operation for non-specific reactions was carried out in the evening, and the mixture was further diluted 2 times with PBS. ] and reacted with shaking at room temperature. After 2 hours, after washing the replica filter three times for 15 minutes each time with PBS-T (PBS solution containing 005% Tween 20), peroxidase-labeled anti-human IgG and IgM goat antibody (MB
The cells were immersed in an incubation buffer (PBS solution containing 1% bovine serum albumin) containing Fab. 1 hour later
PBST for 15 minutes each time, 4 times in total, then PBS
After washing for 5 minutes with
It was immersed in 2O2 to develop color.

Phage corresponding to the plaque developed on the NC filter were selected and subjected to secondary screening. That is, 200 PFU of each phage selected in the secondary screening was separately infected with Escherichia coli Y1090 together with 200 PFU of a phage without an inserted fragment, and transferred to an LB plate in a 995m petri dish (manufactured by Becton Dickinson) to prepare a replica filter. These were screened for antibodies using the method described above, and phages that react with NANBI (convalescent and carrier phase chimpanzee plasma with good reproducibility) were identified.
A clone (CI(-15)) was obtained.

For the clone obtained in (4), create a replica filter in the same way as the secondary screening in (4) and C, and
NBH recovery phase, carrier phase and normal chimpanzee serum or DI after ammonium sulfate precipitation! A plaque assay was performed using an IgG fraction purified with AE-Cellulofine Lamb (manufactured by Seikagaku Corporation). The method is the same as that for antibody screening, but when using chimpanzee IgG fraction for the next antibody reaction, PB is added to a concentration of 50μ9/1.
Dilute with S, add 1,720 volumes of E. coli lysate, and add 4
It was used after being subjected to absorption treatment for non-specific reactions at ℃ overnight.

As a result of the plaque assay, CH-15 reacted at a high rate with NANBI (carrier stage chimpanzee serum or IgG fraction, but did not react at all with normal chimpanzee serum or IgG fraction).

From this result, CEI-45 is particularly
The phage DNA of CH-15, which can be said to be a clone with high specificity for carrier phase chimpanzee serum, was purified [Experimental Medicine Special Issue, Genetic Engineering Collection Volume 11, P31-32 (
1987)] was cut with the restriction enzyme EcoRI (manufactured by Toyobo Co., Ltd.), and then inserted into the BcoR1 site of the p[Ic118 vector to perform subcloning [Douglas
Hanahanj, Mo1. Biol.

■ Island P557-580 (1983)], this subcloned plasmid pCH-15 was
After the cleavage, electrophoresis was performed on a 1.5% agarose gel, and an approximately 380 bp insert fragment (CI(, -15)) was confirmed (Figure 1).

Southern blot analysis using CI (-15) was performed as described below.
Chromosomal DNA was purified from the liver of the F strain infected with NANB [(acute phase (8 weeks after inoculation with NANBFI virus), and also from the white blood cells of a normal person. After cutting 1 to 20 Jtg each with EcoRI, electrophoresis was performed on a 2% agarose gel. Expand, NC
Transferred to filter. This filter was subjected to Southern hybridization using a [lp]-labeled Cl-15 probe using the multiprime method (Fig. 2). As can be seen from this figure, when the CI (-15 probe was autoradiographed for - weeks), the CI (-15 probe) was
Although it reacted with the H-15 clone, it did not react with normal and NAIH acute phase chimpanzee chromosomal DNA or normal human chromosomal DNA. From this, C
H-15 is not a clone derived from human chromosomal DNA, but is thought to be derived from a foreign nucleic acid such as a virus.

CH- (A) Determination of base sequence of C1(-15 clone CH-
Plasmid DNA incorporating 45 gene fragments was used as a template, and [α-”P]dCTP (800Ci/-m
ol) was used in the reaction. Klenow frag■en
The polymerase reaction using t was carried out using Takara Shuzo's 7DEAZ^ sequencing kit. Using 8% polyacrylamide-8-urea gel, electrophoresis was carried out at 1800 V for 4 hours, followed by exposure for 16 hours.

B r - The results of decoding the base sequence obtained above and the amino acid sequence predicted therefrom are shown in FIGS. 3 and 4, respectively.

As a result of searching the obtained nucleotide sequence and amino acid sequence in the database (described above), no virus, bacteria, or other substances showing high homology were found. In addition, as a result of comparison with the nucleotide sequence and amino acid sequence of the non-Allf hepatitis B virus announced by Chiron, it has 83.1% homology with the nucleotide sequence of C15 clone and 89.5% homology with the amino acid sequence. The region was present in the sequence published by Chiron (see Figures 5 and 6).

(8) Based on the amino acid sequence encoded by B CFI5 in the antibody assay using 29er and 37wrer, the following 29
A synthetic peptide consisting of amino acids (29aer) and a synthetic peptide consisting of 37 amino acids (37aer) were synthesized.

29aer: Glu Phe Asp Glu Met Glu G
lu Cys^Ia Ser Old 5Leu Pro Ty
r Val Glu Gln Gly Met Gln
Leu Ala Glu Gln Phe Lys G
ln Lys^1a37+11er: Pro^sp Arg Glu Val Leu Ty
r Arg Glu PheAsp Glu Met
Glu Glu Cys Ala Ser His L
euPro Tyr Val Glu Gin Gly
Met Gin Leu AlaGlu Gln P
He Lys Gln Lys Ala was synthesized using a 43OA peptide synthesizer manufactured by Abride Biosystems, and purified using HP L for synthetic peptide purification.
C column (Aquapore Preplo, C-8
,3QOA pore 5ize, 20um 5p
herical 5illJ:a, 10 years IDX
250-1, Applied Biosystems, Inc.) 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 PBS
), add 200 IA to each well of the immunoplate, react overnight at 4°C, and add 200 IA to each well of the immunoplate.
Wash with PBS (PBS4) containing 1% Tween 80, 0.1% BSA (bovine serum albumin), PBS
Coating was done with 250 d/well of solution. Thereafter, 200 Δ of 01% BSA PBS-T was added to each well, 20 Δ of plasma samples were added thereto, and after stirring gently with a plate mixer, the mixture was allowed to react at 37° C. for 1 hour.

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

After washing with PBS-T, TMBZ (3', 3', 5',
5°.

After adding 200 J of a solution of tetramethylbenzidine hydrochloride) f to each well and reacting at 37°C for 30 minutes, kojic acid was added at 50 β/well to stop the reaction. This was measured using a microplate reader with two wavelengths of 450n and 650n.

As a result, we performed the above immunosandwich assay on 165 human plasma samples that were thought to be blood infected with non-A, non-B hepatitis virus. showed positive for
In the measurement method using 39aer, 13 out of 165 samples
Five samples showed positive results. That is, the non-A, non-B hepatitis virus antibody measurement method using 39aer of the present invention is a measurement method with an extremely high detection rate.

The non-A, non-B hepatitis virus antibody measurement method using the peptide of the present invention is an extremely superior measurement method with less non-specific reactions compared to conventional methods.

[Brief explanation of the drawing]

Figure 1 shows pCH (5) cloned in the present invention.
FIG. 2 is a schematic diagram of the EcoRI insert fragment of 1.5% agarose gel electrophoresis after opening. Figure 2 shows CH-15 cloned in the present invention.
FIG. 2 is a schematic diagram of Zazan hybridization between human and chimpanzee chromosomal DNA. FIG. 3 shows the non-A1 cloned according to the present invention! - Shows the base sequence of a nucleic acid fragment encoding a hepatitis B virus antigen. FIG. 4 shows the amino acid sequence encoded by the non-A1 and B hepatitis virus nucleic acid fragment cloned in the present invention. Figure 5 compares the nucleotide sequence of the non-A IP-B hepatitis virus (1-ICV) gene published by Chiron and the nucleotide sequence of CI (-45 clone).
■ Genome nucleotide sequence. The bottom row shows the nucleotide sequence of CH-15 of the present invention, with common nucleotides indicated by cars. Figure 6 shows the non-A, non-B hepatitis virus published by Chiron.
HCV) gene-encoded amino acid sequence and CFI-1
This is a comparison of the amino acid sequences of 5 clones. The upper row is the amino acid sequence encoded by the HCV genome, and the lower row is the amino acid sequence encoded by C[(-15) of the present invention.5 Common amino acids are shown in the book.

Claims (15)

[Claims] (1) Non-A containing the following amino acid sequence or a part thereof
Non-B hepatitis virus antigen peptide. [There is a gene sequence] (2) the first (1) comprising at least the following 29 amino acids;
) The non-A, non-B hepatitis virus antigen peptide described in item 1. [There is a gene sequence] (3) Said (1st) comprising at least the following 37 amino acids:
) The non-A, non-B hepatitis virus antigen peptide described in item 1. [There is a gene sequence] (4) The non-A, non-B hepatitis virus antigen peptide according to item (1) above, wherein the peptide is an expression product obtained by genetic recombination technology. (5) The non-A, non-B hepatitis virus antigen peptide according to item (1) above, wherein the peptide is a chemically synthesized peptide. (6) The non-A, non-B hepatitis virus antigen peptide according to item (5) above, which consists of the following amino acids. [There is a gene sequence] [There is a gene sequence] (7) The non-A, non-B hepatitis virus antigen peptide according to item (5) above, which consists of the following amino acids. [There is a gene sequence] (8) Detection of non-A, non-B hepatitis-related antibodies, characterized in that the peptide according to any one of items (1) to (7) above is used as an antigen, and antibodies specific to the peptide are detected. Method. (9) A nucleic acid fragment containing a nucleic acid sequence encoding a peptide consisting of the following amino acid sequence or a portion thereof. [There is a gene sequence] (10) The nucleic acid fragment according to item (8) above, which comprises at least a nucleic acid sequence encoding a peptide consisting of the following 37 amino acids. [There is a gene sequence] (11) The nucleic acid fragment according to item (9) above, which consists of the following nucleic acid sequence or a part thereof. [There is a gene sequence] (12) A nucleic acid probe for detecting a non-A, non-B hepatitis virus gene, characterized in that it consists of a nucleic acid fragment of at least 10 bases contained in the following base sequence. [There is a gene sequence] (13) A method for detecting non-A, non-B hepatitis virus, which comprises hybridizing the nucleic acid probe of item (12) with the nucleic acid of a target sample. (14) An anti-non-A, non-B hepatitis virus antibody prepared using the peptide described in item (1) above as an antigen. (15) An immunological detection method for non-A, non-B hepatitis virus, which comprises using the antibody according to item (14) above.