JPH03228681A - Nucleic acid fragment coding non-a non-b type hepatitis virus antigen peptide and method for utilizing same fragment - Google Patents

Abstract

PURPOSE:To enable detection of non-A non-B type hepatitis virus in lever structure by cloning a causative virus (gene) of non-A non-B type hepatitis virus. CONSTITUTION:A plasma obtained by adding a precipitating agent such as polyethylene glycol to a human plasma and ultracentrifuging the plasma and concentrated to about 1000 times is treated with guanidium thioacetate and extracted with phenol/chloroform and precipitated with ethanol to purify total nucleic acid in concentrated plasma. Then cDNA synthesized from DNA obtained by decomposing and purifying human- derived DNA containing deoxyribonuclease is inserted into lambdagt11 vector to prepare cDNA library. Then replica obtained by culturing Escherichia coli infected by lambda phage and then culturing the phage infected Escherichia coli using a nitrocellulose filter is reacted with a human or chimpanzee serum at recovery stage or acute stage of non-A non-B type hepatitis and then reacted with enzyme labeled antihuman IgG or IgM and reacted with a substrate solution to color plague and a phage corresponding to the colored plague is selected and subjected to secondary screening to provide the nucleic acid fragment coding non-A non-B type hepatitis virus antigen peptide.

Description

Detailed Description of the Invention The present invention provides a gene fragment encoding a viral antigen of a virus that causes serotype hepatitis that is neither A nor B (non-A, non-B hepatitis virus), and a non-A, non-B hepatitis virus antigen. Concerning peptides and their uses.

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 the hepatitis A virus is an RNA virus with a diameter of 27 nm that belongs to the picornavirus [Finest
on, S.

M, et al,, 5science 182 p1
026 (1973)], on the other hand, the hepatitis B virus was found to be a DNA virus with a straight 1142n-sized envelope that belongs to the hepadnaviruses. [
Dane, 0. S., et al.

Lancet, + p695 (1970)] Furthermore, immunoserological diagnostic methods for these hepatitis viruses have now been established.

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

M., 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 it has not been reduced to zero, and furthermore, patients with hepatitis who have developed
No evidence of hepatitis B or hepatitis B infection was found. 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, 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, it has become clear that there is a second type of viral non-A, non-B hepatitis that is prevalent through oral infection in India, Burma, Afghanistan, and Africa [
Khuroo, M., S., A., J., Med.
, .

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 Annual Meeting of the Japanese Society of Hepatology, p. 151 (1989) ].

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. The possibility of herpes, cytomegalo, or Epstein-Barr virus infection was ruled out, and drug-induced, alcohol-induced, or autoimmune hepatitis was ruled out, and the patient was diagnosed with non-A, non-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, 1 p463 (197
8)] Despite many efforts around the world, the causative virus is still unknown even after more than 10 years. Type A and B reactions, immunoelectric countercurrent method, radioimmunoassay, fluorescent antibody method, electron microscopy, etc.
Almost every approach used in hepatitis research has searched for viruses and related antigen-antibody systems, but nothing is certain.

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 [Pr1nce, A. M.,
Ann, Rev. Microbiol, 37
．． p217. (1983)].

A recent example is the retrovirus theory by 5eto et al. [5eto, B, et &+,: La
ncet, I p941-943 (198
According to them, reverse transcriptase activity was detected in serum and blood products that have been shown to cause non-A, non-B hepatitis in chimpanzees, and in sucrose density gradient centrifugation, this enzyme was detected in 1. 14g/■! In other words, it had a buoyancy density similar to that of retroviruses. Subsequently, Pr1nce et al. reported that retrovirus-like particles were observed when chimpanzee liver primary culture *S+ cells were inoculated with patient serum [Pr1nce, A, M, et i
f, Lancet, I: p1071-1075 (
1984)], while reverse transcriptase activity is Ho
This was rejected by a follow-up study by Llinger et al. [Bolli
Lancet.

I p41 (1986) 1. Furthermore, Pr1nce
The virus particles observed by et al. were ruled out as myxovirus contamination.

Problems that make research on non-A, non-B hepatitis difficult are that the virus concentration in serum is as low as 10 to 10, and that some chimpanzees have been reinfected with the same inoculum, so the presence of antibodies is doubtful. The problem is that 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 [Ch.
oo, Q et al, 5science, 244
．． P2S5-362 (1989), Kuo, G.
et al., 5science, 244. p362
-364 (1989) ], the properties of the virus itself,
The properties of the virus' constituent proteins have not yet been clarified.

In general, diagnostic methods for different viruses are completely different due to differences in their immunoserological and molecular genetic properties. In addition, differences in strains have some differences in immunoserological properties, so when using the same diagnostic method, differences in detection sensitivity between strains will occur, and in vaccines, differences in immunogenicity and ability to protect against infection will occur.

In molecular genetic diagnostic methods, such as DNA probe diagnosis, it is generally known that hybridization between a probe and a viral nucleic acid is not practical unless the homology at the nucleic acid level is extremely high. That is, there may be cases in which DNA hybridization does not occur due to differences in the nucleic acid level between strains, making DNA probe diagnosis ineffective.

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. Since it is known that there are
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 succeeded in cloning the gene encoding the antigenic peptide sequence of non-B hepatitis virus.

That is, the present inventors used blood donor plasma with high GPT levels to detect non-A, non-B hepatitis viruses using an immunoscreening method that incorporates a new molecular genetic method that is different from conventional immunoserological methods. Furthermore, the expression product obtained by expressing this gene fragment using genetic recombination technology has a protein level that is similar to that of non-A IF and B hepatitis patient serum. It was confirmed that it reacts specifically, and the present invention was completed.

When cloning nucleic acid fragments as is the object of the present invention, livers of Japanese people infected with non-A, non-B hepatitis and livers of chimpanzees infected with non-A, non-B hepatitis are used as research materials. It is conceivable to use the liver to extract mRNA and synthesize cDNA, from which virus-specific cDNA is selected 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. In humans, the existence of carriers of non-A, non-B hepatitis has been confirmed, and the higher the donor's GPT value during blood transfusion, the higher the incidence of non-A, non-B hepatitis after transfusion. It is estimated that the frequency 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, plasma from a chimpanzee that has been inoculated with the serum of a Japanese non-A11-B hepatitis patient and has developed non-A, non-B hepatitis can also be used, but there are currently some problems due to the availability of chimpanzees.

As mentioned above, the concentration of non-A, non-B hepatitis virus in plasma is estimated to be only about 10 to 10", so extraction of viral nucleic acid and synthesis of cDNA requires 10"
It is necessary to concentrate the 9 viruses by 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 (PI!G), which we used, is a mild method that can be performed relatively easily, is suitable for processing large amounts of plasma, and is less likely to inactivate the virus. be.

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

The plasma, which has been reduced approximately 1000 times in this way, is treated with guanidinium thiocyanate, extracted with phenol/chloroform, and purified with ethanol precipitation to purify all the nucleic acids in the concentrated plasma. Human-derived DNA is degraded, and RNA is purified by phenol/chloroform extraction and ethanol precipitation.

cDNA is synthesized from the purified RNA and inserted into the λgtl 1 vector to create a cDNA library.

E. coli is infected with the λ phage, spread on a bacterial culture plate, and cultured at 42°C for several hours. After that, cover with a nitrocellulose filter (NC filter) and culture for several hours.
Peel off the NC filter and take a replica.

This replica is treated with a blocking solution, washed 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 90b was analyzed by acrylamide gel electrophoresis.
The insertion fragment of p (jnhl-1) 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 agarose electrophoresis, $2p-labeled jnhl-
Southern hybridization was performed using one clone. jnhl-1 did not react with any DNA, and therefore it was determined that jnhl-1 was not chromosomal-derived DNA.

In addition, a pool of Japanese GOT and GPT-high plasma (non-A
Non-B hepatitis infectivity has been confirmed in chimpanzee infection experiments), non-Al of the American NIH-derived F strain): RNA was extracted from chimpanzee plasma passaged with hepatitis B and Japanese normal human plasma, and cDNA was extracted. A PCR reaction was performed using part of the nucleotide sequence of the jnhl-1 clone as a primer [5aiki et al.

5science 239. p487- (1988)
] was carried out. Similarly, DNA was extracted from normal human liver and a PCR reaction was performed using the same primers. the result,
Japanese GOT, from a pool of high GPT plasma only
h'! -1 base sequence was detected. This suggests that the non-A, non-B hepatitis virus that we captured that contains the Jnhl-1 nucleotide sequence is considerably different in its nucleic acid sequence from the non-A, non-B hepatitis virus of strain F derived from the US NIH. ing.

The DNA sequence of the Jnhl-1 clone of the present invention was determined by the dideoxy method. As a result, the jnhl-1 clone is a total of 80 bp cDNA fragment derived from the non-A, non-B hepatitis virus gene, and its base sequence is the 35th! It was as shown in I. This nucleotide sequence and the amino acid sequence deduced from it are stored in a database (Genetyx-C
D Software Development 1989)
Until now, there were no known viruses, bacteria, or anything else that showed any homology.

From this amino acid sequence, the hydrophilicity/hydrophobicity pattern of the peptide encoded by jnhl-1 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 highly hydrophilic peptide as a whole.

In this way, the cDNA fragment obtained in the present invention is non-A
11- The fact that the hepatitis B virus antigen encodes a highly hydrophilic peptide region was considered to be very significant from an immunological standpoint. Moreover, since such hydrophilic peptides are easy to handle, they are very useful from a practical standpoint.

In order to further confirm the relationship with non-A, non-B hepatitis, plaque immunoassay and dot immunoassay for JNHL-1 were performed using serum from a large number of hepatitis patients and normal individuals. As a result, a higher proportion of non-A, non-B hepatitis patients were detected to be antibody-positive than in normal subjects, hepatitis B, and other hepatitis groups, and immunoassay showed that non-A, non-A, non-B hepatitis was detected at the protein level.
#Proven specificity for hepatitis B.

Since multiple factors are thought to be involved in non-A, non-B hepatitis, we next synthesized primers based on part of the base sequence of jnhl-1 and performed a PCR reaction on human pooled plasma with high GOT-GPT levels. and subtype cloning was carried out. After inserting the PCR reaction product into the λgtll vector and performing 1nvitro packaging to prepare an infectious phage solution, screening with antibodies,
Alternatively, hybridization was performed using an oligo probe in jnhll. As a result, two clones of phages (jnhl-13, jnhl-1
6>, 4 clones of phages that react with the oligo probe (jnhl-2, jnhl-4, jnhl, jnhl
-6) was obtained. The nucleotide sequences of these six clones were similarly determined by the dideoxy method (see Figure 3), and the deduced amino acid sequences were compared (see Figure 4).

The gene sequence of the present invention can be expressed using an appropriate expression system and used for antibody testing of non-A, non-B hepatitis viruses, or can be used to immunize animals with the expressed protein to generate antibodies. It is also possible to detect non-A, non-B hepatitis virus in the liver tissues of patients infected with non-A, non-B hepatitis.

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

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

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

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

HBs antigen negative 6PT donated by the Japanese Red Cross Society
Human pooled plasma (8,5111>) with a value of 100 or more was concentrated 1000 times by the following method. First, the human pooled plasma was roughly centrifuged to remove insoluble matter.
M sodium chloride solution, then 40% (W/10 volume)
W) A polyethylene glycol solution (PEG6000, manufactured by Wako Junkei Co., Ltd., average molecular weight 7500) was added at 4°C with stirring. -After standing still for an hour, rotate at 7000 rpm.
Centrifuge for 20 minutes, remove the supernatant, and add THE solution (10mM Tr 1s-HCl
, pH 7,4, 1mM EDTA, 140mM Nac
1) was added and redissolved. Add this solution to 20% of the adhesions,
15%, 10%, and 5% TNE solutions were layered on top of a centrifuge tube in stages, and the mixture was heated at 4°C and 80,000X G for 1 hour.
After 2 hours of ultracentrifugation, the supernatant was removed, and the precipitate was dissolved in 8 ml of PBS to obtain a 1000-fold concentrate of Gffr and GPT-high human boulevard plasma.

First, 5 times the amount of guanidium thiocyanate solution (4M guanidium thiocyanate, 50mM Trls-HCI pH 7,6,
10mM EIITA, 0.1M 2-mercaptoethanol, 2% sarcosyl) was added, stirred, extracted with phenol/chloroform, and purified by ethanol precipitation using glycogen as a carrier.Next, the total nucleic acids in the concentrated plasma were purified. In order to degrade human-derived DNA present in
．． 15KU/ml (Boehringer/Mannheim),
50ml Tris-HCI pH7,4,1 intestinal MED
3 in a mixed solution of 400A of TA, 101M MgC1z.
It was treated at 7°C for 30 minutes. After that, 250 Oboro MEDTA
The reaction was stopped by adding 16 ug of the solution and 10% SDS solution 8IA, 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 impurities that are thought to be present in trace amounts, QIAGIEN pack-100 (
The purification operation was performed using DIAGEN (manufactured by DIAGEN).

cDNA--+- All the RNA purified by the above method was converted into cDNA using cDNA synthesis system plus (manufactured by Amajam).
After the synthesis, the synthesized cDNA was converted to λgtl using cDN^ cloning λgtll (manufactured by Amajam).
1 vector. In vitro packaging resulted in a library of 1.8X 10' plaque forming units (PFU).

(4) Since the primary antibody used for screening the non-A, non-B hepatitis (NANBH) convalescent and carrier cDNA library was chimpanzee plasma from the FIANBH convalescent and carrier stages, a high nonspecific reaction was expected. Therefore, in order to suppress this non-specific reaction, E. coli Y10, which is used for the absorption operation of chimpanzee plasma for screening,
Ninety lysates were prepared. That is, from a single colony, LB medium [1% Ba
cto-trytone (manufactured by Difco), 0.5% B
20 ml of E. coli Y1090 culture solution cultured overnight at 37°C in acto-yeast extract (manufactured by Difco, 1% NaCl, pH 7.5) was added to 2 g of LB medium, and further cultured overnight at 37°C. This culture solution was transferred to a centrifuge tube and centrifuged at 9000 rpm for 10 minutes at 4°C, and the supernatant was removed to obtain a precipitate. 41 per gram of this sediment
of RIPA solution (1% sodium deoxycholate, 1%
Triton X-100, 0,31! NaC1,O,
L%SDS, 0. IM Tris-FICI p
Add H7,5,1ml PMSF> to solubilize,
This was further centrifuged at 9,000 rpm for 10 minutes at 4°C, and the supernatant was used as Escherichia coli lye zate.

B Spruce--2゛Ple-Gffr
, - LB plates [
1.5% Agar (manufactured by Hinaga Pharmaceutical Co., Ltd.), 1% Bacto
-tryptone, 0.5% Bacto-yea
st extract, 1% NaClpFI7.5.5
Bacterial culture plate containing 0 laps/ml ampicillin (
Manufactured by Nunc; 23C × 23C intestine)] 100OOP per unit
Take the FU phage and inject it into E. coli Y1090 at 37°C.
Infect for 5 minutes and add Top Agar 40ml (0
, 7% Agar, 1% Bact.

tryptone = 0.5% Bacto-yeas
t extract, 1%N&C11pF17.5
．． 50μ9/Ken1 ampicillin) and cultured at 42°C for 4 to 5 hours. After that, a nitrocellulose filter impregnated with 10■MIPTG (manufactured by Sigma) (
NC filter: Manufactured by S&S, Code B^85
．． 23 cm x 23 cm) was covered, and the 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%).
(PBS solution containing 0.05% Naps) and shaken overnight at 4°C.

A replica filter soaked overnight in C 1-nin blocking solution was
After washing with BS, NANBF1 was diluted 10 times with PBS.
Convalescent and carrier phase chimpanzee pool plasma (screening plasma) [NANBH Convalescent and carrier phase chimpanzee pool plasma was diluted 5 times with PBS, added with 1/20 volume of E. coli lysate, and subjected to non-specific reaction at 4°C overnight. This was followed by an absorption operation and further diluted 2 times with PBS. ] and reacted with shaking at room temperature. 2
After an hour, the replica filter was washed three times with PBS-T (PBS solution containing 0.05% Tween 20) for 15 minutes each time, and peroxidase-labeled anti-human IgG and IgM goat antibodies diluted 1000 times each ( )I
The cells were immersed in an incubation buffer (PBS solution containing 1% bovine serum albumin) containing Fab, manufactured by BL, and reacted at 37° C. with shaking. After closing at 1 o'clock, PB
ST for 15 minutes each time, 4 times in total, then PBS for 5 times.
After washing for minutes, color developing solution [0.02% DAB (manufactured by Sigma), 0.1% NIC1&.6H20, 0.005% [2
02] to develop color.

Phage corresponding to the plaques developed on the NC filter were selected and subjected to secondary screening. That is, 200 PFO of each phage selected in the second screening was separately incubated with E. coli Y along with 200 PFU of a phage without an insert.
1090 and re-spread on a LB plate in a 590 Petri dish (manufactured by Becton Dickinson) to prepare a replica filter. These were screened for antibodies using the method described above, and 1 phage that reacted with reproducible chimpanzee plasma in the convalescent and carrier stages of Page ANBl'1 was selected.
A clone (Jnhl) 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 DEAE-cellulofine serum after ammonium sulfate precipitation (
Plaque assay was performed using the IgG fraction purified by Seikagaku Kogyo Co., Ltd.). The method is the same as that for antibody screening, except that chimpanzee I
When using gG chicken, add P to a concentration of 50Itg/ml.
Dilute with BS, add 1720 volumes of E. coli lysate,
It was used after being subjected to absorption treatment for non-specific reactions at 4°C overnight.

As a result of plaque assay, Jnhl reacted at a high rate with NANBH carrier stage chimpanzee serum or IgG fraction, and did not react at all with normal chimpanzee serum or IgG fraction.

From this result, it can be said that johl is a clone with high specificity for chimpanzee serum in the NANBH carrier stage.
After cutting with the restriction enzyme EeoRI (manufactured by Toyobo Co., Ltd.), it was inserted into the Eco11 site of the prJc118 vector, and subcloning was performed [Douglas
Hanahan, J., Mo1. Biol.

IH, P2S5-580 (1983)], this subcloned plasmid pJnhl-1 was
After 1 cleavage, electrophoresis was performed on a 5% acrylamide gel, and an approximately 90 bp insert fragment (jnhl-1) was confirmed (Fig. 1).

Southern blot analysis using jnhl-1 was performed as described below. Normal chimpanzee and US NIB-derived F
Chromosomal DNA was determined from the liver of strain-infected NANBH acute phase (8 weeks after inoculation with NA?IBH virus) and from the white blood cells of normal individuals.
A was purified, 20ttg of each was cut with EcoRI, developed on a 2% agarose gel by electrophoresis, and transferred to an NC filter. This filter is processed using the multi-prime method [3
Southern hybridization was performed using the jnhl-1 probe labeled with [2p] (Fig. 2). As can be seen from this figure, the jnhl-1 probe was autoradiographed for -weeks, and the jnhl-1 probe was compared with the Jnhl-1 probe before subcloning.
Although it reacted with clones, it did not react with normal and NANBH acute phase chimpanzee chromosomal DNA or normal human chromosomal DNA. From this, jnhl
It is considered that -1 is not a clone derived from human chromosomal DNA, but is derived from an exogenous nucleic acid such as a virus.

Plasmid DN incorporating the jnhl-1 gene fragment
A is used as a template, [a-82P] dCTP (80
0C1/mmol) was used in the reaction. Xle
Polymerase reaction using now fragment was carried out using Takara Shuzo's 7DEAZ Sequencing Kit. Using 8% polyacrylamide-8M urea gel, electrophoresis was performed at 1800 V for 4 hours, followed by interlock exposure at 16 o'clock.

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

The phylophilicity/hydrophobicity profile of the predicted amino acid sequence of jnhl-1 is shown in FIG.

As a result of searching the obtained nucleotide sequence and amino acid sequence in the database (described above), no viruses, bacteria, or other substances showing high homology were found.

Gff was used as the material for obtaining the jnhl-1 clone.
r.

GPT-high human boule plasma, NAN from US NIFI
Using a PCR reaction, Jnhl was detected using the plasma of a chimpanzee that had become chronic after being inoculated with the F strain of BH (infectivity has been confirmed), normal human plasma, and chromosome 11NA derived from human liver.
-1 base sequence was detected. First, 1 liter of each plasma was precipitated using a 5M sodium chloride solution and a 40% (w/w) polyethylene glycol solution in the same manner as in (1), and 500-guanidine was added to the precipitate. Thiocyanate solution was added, and total nucleic acids were purified by phenol/chloroform extraction and ethanol precipitation. this,
50mM Tris-HCI pH8,3, 61M
MgCh, 40mM KCl, 1mM IITr, ld
dNTPs, 1.3Xtl/ml RNasin, 3
Reaction was carried out at 37° C. for 1 hour and 30 minutes in a mixture of 0 mg/■1 random primer and 4 KU/ml reverse transcriptase (manufactured by BRL) in 2OIA. Take l- of this reaction solution and make 10mM
Trls-HCI pH8, 3, 50mM KCI,
1.5m14 MgCh, 0.01% (w/v) gelatin, 1100n dNTPs, 250nM primer (positions shown in Figure 6) 20σ/+*I Taq
In a mixed solution of polymerase, 94°C; 3
0 seconds, 55°C; 3θ seconds, 72°C; The reaction was carried out for 40 cycles, with 1 minute being one cycle (using a thermal cycler manufactured by Perkin-Elmer Cetus).

For chromosomal DNA derived from human liver, 10Mg
PCR reaction was carried out under the same conditions as above in a reaction solution having the above composition. After the PCB reaction, each sample was taken and developed on a 2% agarose gel by electrophoresis, and then subjected to NC
Transferred to filter. Add this filter to ['allp
Hybridization was performed using a J-labeled oligo probe within JNHL-1 (the position is shown in Figure 6). As a result, the JNHL-1 base sequence was determined from the Gffr and GPT-high human pooled plasma that served as the material. was detected in normal human plasma, chimpanzee plasma of the F strain derived from the U.S. NIH, and chromosome DtlA (Figure 8). After transformation of E. coli JM109, dot immunoassay was performed.

Since expression of this expression plasmid is induced by temperature shift from 30°C to 42°C, the following method was used.

The transformed E. coli was transformed with ampicillin (Ap) containing (50
Mg/d) LB was cultured at 30°C overnight, and the next day, after dilution with LB so that the Klett value was 80, the cells were transferred to 30°C for 1.5 hours, and then transferred to 42°C and cultured for 2 hours to induce expression.

After that, the bacteria were collected and dissolved in 1wdPBS-T, 1g of glass beads were added and crushed with a portex mixer, and the pellet was mixed with 1-50M TrisHCl, p
It was suspended in H8, 0, 10mM EDTA and used for dot assay. This suspension was spotted into a 5Δ spot on a nitrocellulose filter. After drying, the steps from blocking reaction to coloring reaction were carried out in the same manner as in (4)C. The results are shown in Table 1.

(Left below) Table Serum positivity/sample positivity rate (X) Normal person 0/20 0.0 Non-A, non-B
Hepatitis patient 17/30 56.7 Hepatitis B patient
1/9 11.0 Other hepatitis patients 0
/10 0.0 or more, the positive rate is extremely high at 56.7% in the non-A, non-B hepatitis patient group, whereas in normal people, hepatitis B patients, and other hepatitis patients, the positive rate is very high at 56.7%. The positive rates were extremely low at 0.0%, 11.0%, and 0.0%, indicating that the jnhl-1 clone of the present invention is specific for non-A, non-B hepatitis.

Since multiple factors are thought to be involved in non-A11-B hepatitis, a primer (see Figure 7) was synthesized based on a part of the nucleotide sequence of jnhl-1, and this was used to infect jnhl-1. (GOT-GP which became the material for cloning
PCR reaction was performed on T-high human pooled plasma (derived from Japanese people) and cloning was performed. The PCR reaction conditions are (
This was carried out according to the method described in 8). The PCR reaction product was cloned into the λgtll vector using cDNA cloning λgtll (manufactured by Amar-Jam),
in vitr.

After packaging and preparing an infectious phage solution, prepare a replica filter for screening according to the method in (4) B and use it for antibody screening or mixed oligo probes in jnhl-1 (the position of which is shown in Figure 7). Screening was carried out by hybridization using (shown below).

The same procedure as in the secondary screening of (4)C was carried out using carrier phase chimpanzee pool plasma as the antibody screening plasma. As a result of screening, 4 clones (jnhl-
2, Jnhl-4, jnhl-5, jnhl-6), phage that reacts with carrier-phase chimpanzee plasma.
Clones (jnhl-8, jnhl-16) were obtained. The nucleotide sequences and amino acid sequences of these six clones are shown in FIGS. 3 and 4.

[Brief explanation of drawings]

Figure 1 shows pjnhl cloned in the present invention.
It is a schematic diagram of the F, coR1 insertion fragment of -1 after 5% acrylamide gel electrophoresis development. Figure 2 shows jnhl- cloned in the present invention.
FIG. 1 is a schematic diagram of Southern hybridization of No. 1 with human and chimpanzee chromosomal DNA. FIG. 3 shows the base 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. Figure 5 shows Jnhl-1 analyzed based on the amino acid sequence.
shows the hydrophilicity/hydrophobicity profile of the peptide encoded by . FIG. 6 shows the positions of the primers and oligo probes in the HL-1 base sequence used in the PCB reaction in Example (8). FIG. 7 shows the positions and base sequences of mixed primers and mixed oligo probes with reference to the base sequence of jnhl-1 used in the PCR reaction in Example (10). FIG. 8 is a schematic diagram of hybridization of a gene amplified from human chromosomal DNA and nucleic acid in serum using a 1'CR reaction using primers in the base sequence of jnhl-1 cloned in the present invention. It is.

Claims (10)

[Claims] (1) A nucleic acid fragment encoding a non-A, non-B hepatitis virus antigen peptide. (2) The above item (1), wherein the non-A, non-B hepatitis virus peptide is a peptide containing an amino acid sequence selected from the group consisting of the following amino acid sequences (A) to (C) or a portion thereof: Nucleic acid sequence. (A) [There is a nucleic acid sequence] (B) [There is a nucleic acid sequence] (C) [There is a nucleic acid sequence] (3) The above-mentioned (2) comprising a nucleic acid sequence selected from the group consisting of the following nucleic acid sequences (A) to (G) or a part thereof.
Nucleic acid sequences described in Section. (A) [There is a nucleic acid sequence] (B) [There is a nucleic acid sequence] (C) [There is a nucleic acid sequence] (D) [There is a nucleic acid sequence] (E) [There is a nucleic acid sequence] (F) [There is a nucleic acid sequence] There is a sequence] (G) [There is a nucleic acid sequence] (4) A non-A, non-B hepatitis virus antigen peptide comprising an amino acid sequence selected from the group consisting of the following amino acid sequences (A) to (C) or a portion thereof. (A) [There is an amino acid sequence] (B) [There is an amino acid sequence] (C) [There is an amino acid sequence] (5) The non-A, non-B hepatitis virus antigen peptide according to item (4) above, wherein the peptide is a chemically synthesized peptide. (6) The peptide is a peptide obtained by incorporating the nucleic acid fragment of item (1) into an appropriate expression vector and expressing this in a host cell. Hepatitis B virus antigen peptide. (7) A nucleic acid probe for detecting a non-A, non-B hepatitis virus gene, comprising a nucleic acid fragment of at least 10 bases contained in any of the following base sequences (A) to (G). (A) [There is a base sequence] (B) [There is a base sequence] (C) [There is a base sequence] (D) [There is a base sequence] (E) [There is a base sequence] (F) [There is a base sequence] There is a sequence] (G) [There is a base sequence] (8) A method for detecting non-A, non-B hepatitis virus, which comprises hybridizing the nucleic acid probe of item (7) with DNA of a target sample. (9) An anti-non-A, non-B hepatitis virus antibody prepared using the peptide described in item (4) above as an antigen. (10) A method for immunological detection of non-A, non-B hepatitis virus, which comprises using the antibody described in item (9) above.