JP3269642B2 - Non-A non-B hepatitis virus gene cDNA and antigen polypeptide - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

The present invention relates to a non-A non-B hepatitis virus gene cDNA and a non-A non-B hepatitis virus antigen polypeptide. More specifically, non-A non-A useful for the production of non-A non-B hepatitis virus antigen polypeptides
The present invention relates to a non-hepatitis B virus gene cDNA and a non-A non-B hepatitis antigen polypeptide which is an expression product thereof. Non-A non-B hepatitis virus gene cDNA of the present invention
Is useful as a gene encoding a virus antigen of non-A non-B hepatitis and a gene diagnostic agent. In particular, the non-A non-B hepatitis antigen polypeptide of the present invention may be a non-A non-B hepatitis vaccine, immunoglobulin, polyclonal or monoclonal antibody, immunological diagnostic agent, blood transfusion screening agent, and blood transfusion by affinity column chromatography. It is useful for producing NANBV removing agent in blood for use.

[0002]

2. Description of the Related Art [Definition of non-A non-B hepatitis virus]: As is well known, viral hepatitis is a liver disease caused by hepatitis virus infection. , B, and D (delta) hepatitis viruses have been isolated and identified. In addition, hepatitis D virus (delta hepatitis virus) is a defective virus that cannot be multiplied by itself, and its proliferation always requires hepatitis B virus as a helper virus, and therefore exists only in hepatitis B patients. However, in 1974, type A and B
It has been reported for the first time that there are many cases of viral hepatitis that are not caused by both infections of each hepatitis virus. Since then, research on this has been energetically advanced in various parts of the world, and as a general term for such hepatitis cases, non-A non-B (non-A, non-B)
The term hepatitis he came to be used. Currently non-A non-B
It is believed that there are several types of hepatitis pathogenic viruses. Non-A, non-B hepatitis virus depends on its route of infection.
It is roughly divided into the following two types: epidemic hepatitis type or oral infection type (waterborne) transmitted through water or food.
ly-trusted non-A, non-
B); and a blood-transmittant type that is mainly transmitted via blood and frequently occurs after blood transfusion.
ed non-A, non-B). With regard to the isolation and identification of such viruses, except for oral infectious non-A non-B hepatitis viruses that have already been virologically confirmed in Africa, India, and Southeast Asia as invasive areas, general approval in virology is required. The virus that obtained it is not yet known. Hereinafter, unless otherwise specified, the term “NANB hepatitis” refers to blood-borne non-A non-B hepatitis, and the term “NANBV”
Means blood-borne non-A non-B hepatitis virus, respectively.

[Current status and issues of research on NANB hepatitis]: N
Studies on the epidemiology, clinical, diagnosis, treatment and prevention of hepatitis ANB have been conducted in comparison with hepatitis NANBV and other hepatitis.
Utilizing knowledge and techniques of virology, diagnostics, histopathology, immunology, molecular biology, etc., it is being promoted around the world [Nippon Medical News, No. 3320, 3-10, 1987
Year; History of Medicine, 151 (13), 735-923,
1989; Hepatobiliary pancreas, 21 (1), 5-113, 199
0; Experimental Medicine, 8 (3), 201-233, 199
0]. For example, hepatitis NANB has been reported as follows.

(A) Epidemiology: The number of patients with NANB hepatitis in Japan is estimated by the Ministry of Health and Welfare to be about 60% (about 720,000) of patients with chronic hepatitis, about 40% (about 100,000) of patients with cirrhosis, And about 40% (approximately 7,000) of liver cancer patients, resulting in 16,000 deaths each year. Also,
In the United States, 150,000 to 300,000 post-transfusion hepatitis cases occur annually,
Ninety percent of the donors are NANB hepatitis, and 1-6% of donors are considered to be NANBV carriers.
In other countries, it is estimated that the incidence and carrier rate are the same or higher than those in the United States and Japan. Therefore, the prevention, early diagnosis and establishment of treatment for NANB hepatitis is extremely important worldwide. is there.

(B) Virology: NA previously reported to date
NBV is a spherical shape having an envelope of about 50 nm in diameter and is considered to be similar to togavirus or flavivirus in virus taxonomy or a completely new type of virus. In addition, in chimpanzee hepatocytes inoculated intravenously with NANB hepatitis patient serum, the presence or absence of formation of cytoplasmic tubular structures, pathological findings of nuclear particle appearance, epidemiological findings, presence or absence of chloroform sensitivity, immunological diagnosis, etc. Based on this, the existence of two or more types of NANBV is estimated (for example, Science, 205 , 197-
200, 1979; Journal of Infec
tious Disease, 148 , 254-26
Microorganism, 5 (5), 463-475, 1
989). In particular, compared to both hepatitis A and B, the amount of NANBV in the blood of the patient is extremely small, for example, Chimpa
nzee Infectious Dose (CID)
Is 10 ⁸ -10 ⁹ CID / ml for the B type, whereas it is 10 ⁴ -10 ⁵ CID / ml for the NANB type (Bradley DW: Research Peel).
rspectives in Post-transf
usion non-A, non-B hepatit
is, in “Infection, Immunit
yand Blood Transfusion ”ed
ited byDodd R. Y. & Barker,
L. F; Liss, Inc. New Y
ork, 81-97, 1985). Furthermore, with regard to non-human infection experimental systems, at present, only chimpanzees that form a typical intracytoplasmic tubular structure in the hepatic cytoplasm due to NANBV infection are known as susceptible animals. In short, supply restrictions and economic constraints on the availability of the
It makes difficult and delays definitive infection experiments, identification, and marker discovery of V. Therefore, various strategies relating to NANBV research have been attempted to overcome such problems. For example, NAN called hepatitis C virus (HCV) is derived from the plasma of chimpanzees with NANB hepatitis.
The BV gene cDNA was cloned (Science,
244 , 359-362, 1989), and further confirmed that an antigen called C-100 obtained by expressing the gene reacts with an antibody in the blood of a patient with hepatitis NANB (S).
science, 244 , 362-364, 1989);
NANB without chimpanzee as in the above example
The NANBV gene cDNA is cloned directly from the plasma of a hepatitis hepatitis patient, and the antigen obtained by expressing such a gene is NA
Confirmed that an antigen-antibody reaction occurs with the antibody of the serum of a patient with hepatitis NB (Gastroenterology Japan)
onica, 24 , 540-544, and 545-5.
48, 1989 and the like are known.

(C) Clinical: In general, hepatitis occurs in epidemic hepatitis and sporadic hepatitis according to the population and frequency of occurrence, and also in acute hepatitis, fulminant hepatitis, subacute hepatitis, and persistent hepatitis based on the severity and stage. , And chronic hepatitis. The incubation period of NANB hepatitis is 2 to 26 weeks, and its initial symptoms are less severe than hepatitis B with fever and malaise, and 70% are non-jaundice, so there is a high probability of being overlooked. But NANB
Hepatitis hepatitis is characterized by its susceptibility to chronicity and a shift to cirrhosis. For example, among NANB hepatitis cases in which an increase in serum aminotransferase activity has been observed, 40-50% of the cases become chronic, and 10-20% of the chronic cases have liver cirrhosis.
Also, 0.5-1% of recipients annually develop cirrhosis without subjective symptoms. Unfortunately, further progress is thought to lead to hepatocellular carcinoma or liver cancer. Therefore, eradication of NANB hepatitis is extremely important worldwide from the viewpoint of public health in order to prevent biohazard associated with not only blood transfusion but also bleeding.

(D) Diagnosis: As described above, NANBV (blood
Liquid transmission type) has not yet been identified and
Is unknown, so the diagnosis of NANB hepatitis
Hepatitis A and B, cytomegalo, EB, chickenpox,
Serum of patients specific to each known inflammable virus such as Lupes
Exclusion diagnosis based on serum antibody titers and liver biopsy
Must rely on histopathological diagnosis (S. Shen
lock, Disease of the river
 and Filiary system,8, 326
-333, Blackwell Scientific
 Publications 1989). And this
In parallel with this, for example, serum enzymes, GPT [glutamic acid
Pyruvate transaminase; also known as: ALT (Alani
Aminotransferase)], GOT [(glutami
Oxaloacetate transaminase; also known as: AST
(Aspartate aminotransferase)], guar
Measurement of the activity of nindeaminase (also known as guanase)
(Hepatobiliary pancreas, Vol. 14, 519-522, 1987);
Abnormality of GPT or GOT activity in serum over time
Diagnostic criteria based on continuation of high levels (Journal of the Japanese Society of Transfusion,31
(4), 316-320, 1985;46,
2635-2638, 1988).
In addition, regarding the immunological diagnosis, as described above, NANBV
At present, it is difficult to separate and identify
DNA of NANBV cDNA isolated using epidemiological knowledge
Antigen and NANB obtained by expressing a loan
Antigen-antibody reaction with serum from patients with hepatitis B
You. For example, as an antigen, the plasma originated from NANB hepatitis
Original NANBV cDNA expression product (European Patent Publication No.
No. 363025), NANB hepatitis-onset chimpanzee blood
HCV cDNA expression product from plasma (European published patent application
No. 318216, or JP-A-2-500880), N
NANBVcDN from ANBV-infected chimpanzee liver
A expression product (EP-A-293274, or
JP-A-64-2576 and JP-A-1-124387)
Etc. are also known, and a measurement method by an antigen-antibody reaction and
RIA (radioimmunoassay), EIA (en
Zym immunoassay) is commonly used. But
The antigenicity of such expression products is poor in common with each other,
For example, the expression product of the HCV cDNA, ie, the C-
The 100 antigen is a primary diagnosis of diffuse hepatitis due to HCV infection
Although it serves as an index, the reaction region exhibiting antigenicity is narrow
Because (microorganisms,5463-475, 1989;
Pancreas,2047-51, 1990; History of Medicine,Fifteen
1871, 1989), NANB hepatitis required for treatment
Or confirmed diagnosis of NANBV infection, chronic hepatitis and acute
It is considered insufficient to judge the course of hepatic hepatitis. Obedience
Therefore, the diagnosis of NANB hepatitis, and the judgment of progress and treatment
More detailed virological and immunological
Exploration is expected.

(E) Treatment and prevention: Recently, the usefulness of α and β interferons in the treatment of chronic NANB hepatitis has been reported (hepato-biliary-pancreas, 20 , 59-64, 19).
90; History of Medicine, 151 , 871-876, 198
9). However, an appropriate dose, administration period, and the like have not yet been determined. -Way, a preventive agent for NANB hepatitis,
For example, for the vaccine, the aforementioned NANBVcDN
A (European Patent Publication No. 363025) and HCV cDNA expression product (European Patent Publication No. 3)
No. 18216) is known as a vaccine antigen. However, in the current situation where NANBV itself has not been determined, an antigen for NANBV vaccine is identified from the above expression products having various antigenic determinants (epitope), and the safety and efficacy of such a specific antigen are clinically determined. In
NANBV requires more detailed test research and longer time.
The vaccine has not yet reached the market.

[0009]

Means for Solving the Problems The inventors of the present invention have conducted intensive studies to overcome the above-mentioned problems, and as a result, have known NANs.
It is superior in reliability to BV cDNA and has the longest chain as compared to known NANBV cDNA, and clones the NANBV gene cDNA having the entire open reading frame region and the non-coding region of the gene. By expressing it, we succeeded in the production and acquisition of NANBV antigens that exhibit excellent antigen-antibody reactions not only with chronic but also acute NANB hepatitis sera. Such success is genuine (a
difficult to isolate NANBV, even though the amount of NANBV in blood or in resected hepatocytes is extremely low, about 1 / 10,000 that of hepatitis A or B, in order to obtain the NANBV gene. Without amplifying NANBV using chimpanzees with unknown stray factors that are believed to be the most reliable fresh material from humans containing authentic NANBV,
Our unique technology of extracting intact or as long as possible long RNA directly from NANBV particles contained in whole blood of NANB hepatitis patients or resected liver strips of liver cancer patients with NANB hepatitis And that the handling and storage of fresh
This is because NBV and its gene have been meticulously manipulated so as not to be cleaved or decomposed by enzymes in body fluids or blood. The R thus prepared from the fresh material of human origin
NA is converted into double-stranded cDNA using reverse transcriptase, DNA polymerase I, or the like, and a cDNA library is obtained. Then, in order to screen the NANBV gene from the cDNA library, each cDNA is separated by lambda g
After insertion into a t11 phage vector and expressing it on phage plaques at high concentrations, the expression product and acute NAN
By repeatedly performing enzyme immunoassay (EIA) using the serum of a hepatitis B convalescent patient and the serum of a chronic hepatitis NANB hepatitis, the NANBV gene cDNA was successfully screened. In this way,
Steady production of NANBV antigen polypeptides purified in large quantities and at low cost without generating biohazard was first observed by expression of the cDNA of the present invention using recombinant DNA technology. The present invention has been completed based on these findings.

[0010] Accordingly, an object of the present invention is to provide a hepatitis NANB virus gene cDNA. It is another object of the present invention to provide a NANB hepatitis virus antigen polypeptide useful as a diagnostic agent for NANB hepatitis and an active ingredient of a vaccine. Further, the present invention relates to NANB
It is intended to provide a method for producing a V antigen polypeptide. Another object of the present invention is to provide a diagnostic agent for NANB hepatitis. Still another object of the present invention is to provide a vaccine against NANB hepatitis. The above and other objects, features and advantages of the present invention will become apparent from the following detailed description and the appended claims, taken in conjunction with the accompanying drawings.

Basically, according to the present invention, at least a part of the entire nucleotide sequence of the non-A non-B hepatitis virus having the nucleotide numbers 1 to 9416 shown in FIGS. A base sequence comprising at least one base sequence selected from the group consisting of a first base sequence and a base sequence complementary to the first base sequence;
There is provided an isolated deoxyribonucleic acid comprising at least one base sequence obtained by replacing two bases by degeneracy of a gene codon.

According to another aspect of the present invention, FIG.
The amino acid sequence containing at least a part of the amino acid sequence encoded by the deoxyribonucleic acid containing the coding region from base number 333 to 9362 of the entire base sequence of the non-A non-B hepatitis virus shown in FIG. An isolated antigenic polypeptide is provided.

In the present invention, unless otherwise specified.
The left and right ends of the deoxyribonucleotide sequence are the 5 'and 3' ends, respectively. Unless otherwise specified, the left and right ends of the amino acid sequence of the peptide are the N-terminal and C-terminal, respectively. The NANBV gene cDNA of the present invention and the NANBV antigen polypeptide as its expression product can be prepared and identified according to the following steps (I) to (VII).

(I) Selection and Collection of NANBV Gene RNA Extraction Materials: NANBV RNA extraction materials include NANBV carriers or human or chimpanzee blood, lymph, ascites, hepatocytes, and NANB-type cells affected by the same. Hepatocellular carcinoma associated with hepatitis or hepatocytes of a liver cancer patient can be used. In particular, the amount of NANBV in chimpanzee-derived material is smaller than that of human,
The use of human-derived materials is desirable because there is a high possibility of contamination by a stray factor that makes it difficult to separate NANBV. Of the blood, lymph, ascites and hepatocytes, blood is the easiest,
It can be obtained in large quantities, for example, blood that cannot be used as transfusion blood can be obtained in large quantities from a blood bank. Such blood can be advantageously used as a material for extracting NANBV gene RNA. When blood is used as a material, the blood is first separated into plasma and red blood cells, and the plasma thus obtained is used as a hepatitis B virus surface antigen (WHO expert commit).
tea on viral hepatitis: Ad
vences in viral hepatiti
s, WHO Technical Report Se
ries, 602 , 28-33, 1977) and the gene DNA of hepatitis B virus (Brechot, C; H
adchoel, M; Scotto, J; Dego
s, F; Channely, P; Trepo, C; Tio
llais, P: Detection of Hepat
itis B virus DNA in river
andserum: a direct apprai
sal of the chronic carrie
r state. Lancet, 2 , 765-768,
1981), and using the plasma as a guideline for the diagnosis of NANB hepatitis, eg, GPT (Wroblewski,
F. & LaDue, J.M. S. : Serum gluta
mic-pyruvictransaminese i
n cardiac and hepaticise
ase, Proc. Soc. Exp. Biol. Me
d. , 91 , 569, 1956. ), GOT, guanase and the like. A step of separating the blood into plasma and red blood cells and testing the plasma is performed on various bloods, and is negative for both the hepatitis B virus surface antigen and the gene cDNA, and the activity of the enzyme is abnormally high. For example, pool plasma having a GPT activity of 35 IU / l or more.

The number of NANB hepatitis virus particles in the blood is
Very low compared to the number of hepatitis B virus particles mentioned above. From the results of infection experiments (Bradley, DW: Res
ear perspectives in pos
t-transfusionnon-A, non-B
heratitis: “Infection, Imm
unity and Blood Transfusi
on "editedby Dodd, RY & Ba
rker, L .; F. , Alan R .; Liss, In
c. , New York, 81-97, 1985), N
Since the number of hepatitis A virus particles is expected to be about 1/10000 of the number of hepatitis B virus particles,
For the extraction of A, it is desirable to use a large amount, for example, about 3 to 101 blood. This fresh whole blood is used as a material for extracting RNA of NANBV from virus particles,
To avoid denaturation of NBV and its gene and cleavage / decomposition of the gene by enzymes, store at 1 to 5 ° C, and prepare RNA of NANBV by the following step (II) within 48 to 72 hours after collecting fresh whole blood. It is desirable to complete. When hepatocytes are used as a material, NANB
Approximately 1 to 3 g of resected liver tissue pieces from non-cancerous and cancerous parts obtained from patients with hepatocellular carcinoma or liver cancer associated with chronic hepatitis
Can be used. Hepatocytes used as material
For example, it is stored frozen at -70 ° C.

(II) Preparation of RNA of NANBV: RNA can be extracted and purified from the material obtained in the above (1) by known means. For example, when using fresh whole blood as a material, about 2-101 of blood is subjected to low-speed centrifugation, a plasma fraction of the supernatant is collected, a virus fraction is purified from this plasma, and then obtained by the following RNA extraction. Provide for purification step.

On the other hand, when liver slices are used as an RNA extraction material for NANBV, a diluent containing about 5 to 30 times the volume of the following RNase inhibitor is added to liver tissue, and then a known solution is added. A liver tissue homogenate is obtained by crushing or grinding the liver tissue using a means such as a homogenizer. In addition, as a diluting liquid, 10-150 m
A known buffer solution of M can be used. Next, this was centrifuged at a low speed to collect the supernatant, and the supernatant was subjected to RN.
A Used as a stock solution for extraction and purification. RNA is extracted and purified by known methods, for example, extraction using a mixture of an RNase inhibitor such as heparin, diethyl pyrocarbonate, guanidine thiocyanate and a surfactant, a chelating agent or a reducing agent that enhances protein denaturation. Method: sucrose, cesium chloride, cesium trichloroacetic acid, ficoll [Ph
armasia Fine Chemicals AB
(Sweden)] as a solute; a fractionation method by density gradient centrifugation; a separation method using an affinity column using a poly A chain at the 3 'end which mRNA has specifically; a specificity for a protein synthesized on a polysome A fractionation method for obtaining mRNA-bound polysomes by immunoprecipitation using an antibody; a phenol extraction method based on the principle of two-phase partitioning; and a precipitation method using polyethylene glycol, dextran sulfate, alcohol, or the like can be used individually or in combination. The above-mentioned extraction and purification steps of RNA of NANBV are performed at pH 3 to avoid irreversible denaturation of RNA.
It is desirable to carry out at 10 to 10.

(III) Preparation of double-stranded cDNA from NANBV RNA: Using the previously obtained NANBV RNA, cDNA can be prepared by a conventional method.
That is, using oligodeoxythymidine and random hexanucleotide primers and reverse transcriptase as primers, using the NANBV RNA as a template to synthesize cDNA complementary thereto, the cDNA complementary to each other and the NANBV RNA To obtain a double strand consisting of
Next, ribonuclease H was allowed to act on this, and the NA
After decomposing the NBV RNA and removing it from the cDNA, a double-stranded cDNA is synthesized by a DNA synthase using the single-stranded cDNA as a template. Double-stranded cDNA synthesis can be performed using a commercially available cDNA synthesis kit, for example, cDNA Synth.
essSystem Plus (registered trademark) [Ame
rsham (UK)], cDNA System
Kit (registered trademark) [Pharmacia LKB (Sweden)], cDNA Synthesis
Kit (registered trademark) [Boehringer Man
nheim GmbH (Germany)] or the like. When the amount of synthesized cDNA is small, conventional PCR (Polymerase Chain) may be used.
Reaction method (Errich, HA):
“PCR Technology, Stockton
Press, 1989).
pliTaq [Perkin Elmer Cetus
(U.S.A.))).

(IV) Preparation of cDNA library:
Using the cDNA prepared in (III), c
Create a DNA library. That is, (III)
A cDNA library is obtained by ligating cDNAs of various lengths prepared in the above to a cloning vector.
Known or commercially available vectors such as phage genes, cosmids, plasmids and animal virus genes can be used as the cloning vector.
When a phage gene or a cosmid is used as a recombinable vector, in order to achieve a high degree of stabilization of the vector into which each cDNA fragment is individually inserted and the appearance rate of a transformant, an in vitro test method is used. After performing packaging and obtaining a vector into which each cDNA has been inserted in the form of recombinant phage particles, a population of these phage particles is subjected to cDNA cloning as a cDNA library. On the other hand, when a plasmid is used as a recombinable vector, each of the above-mentioned cDNA fragments is inserted into a plasmid vector, and these cDNA-inserted vectors are transferred to host cells such as Escherichia coli, Bacillus subtilis, and yeast according to a conventional method. The resulting transformant is subjected to cDNA cloning as a cDNA library. When an animal virus gene is used as a recombinable vector, each of the above cDNA fragments is inserted into a virus gene vector, and each of these recombinant viruses is transfected into a susceptible animal cell according to a conventional method. Proliferate in cells. The thus obtained recombinant virus itself is used as a cDNA library.

As a simple method for preparing the cDNA library, commercially available kits such as a cDNA cloning system Lambda gt10 and Lambda gt11 [Amer
sham (UK), BRL (US), Strata
gene (USA), an in vitro packaging system (Amersham (UK), BRL (USA), Stratagene (USA)) and the like.

(V) NAN from cDNA library
Cloning of BV gene cDNA: In this step, a cDNA clone containing the NANBV gene is obtained. When the cDNA library is a transformant, the transformant is cultured on an agar medium to form a colony. On the other hand, when the cDNA library is a recombinant phage particle or a recombinant virus, the recombinant phage particle or the recombinant virus can be transferred to a known susceptible host cell such as Escherichia coli, Bacillus subtilis, yeast, or animal cell culture. After infection, the cells are cultured to form plaques, or the infected cells are expanded. Under known immunoassays, NANBV antigen was used using acute NANB hepatitis patient convalescent serum, chronic NANB hepatitis patient serum, chimpanzee serum infected with NANBV irrespective of hepatocytoplasmic tubular structure forming or non-forming type. A colony, plaque, or infected cell that specifically produces is selectively isolated by reacting it with at least one of the above-mentioned sera. For a strict selection of colonies, the previous steps were repeated and from each colony, plaque or infected cell selected and isolated, Molecu by Maniatis et al.
lar Cloning, A Laboratory
Manual (Cold Spring Labora)
tory [U. S. A], 309-433, 1982).
To isolate a NANBV gene cDNA clone. The immunoassay can be performed, for example, by an enzyme-labeled antibody technique using an antibody labeled with an enzyme peroxidase or alkaline phosphatase, or a fluorescent antibody technique using an antibody labeled with fluorescein isothiocyanate or europium. In the immunoassay according to the above-described technique, it is desirable to perform the immunoassay by an indirect method that can achieve a highly sensitive immunoassay even when a small amount of patient serum is used. In this indirect method, it is desirable to use the serum of NANB hepatitis patients or NANB hepatitis chimpanzees containing relatively large amounts of antibodies specific to NANBV as the primary antibody, and use enzymes or fluorescent substances as the secondary antibody. Labeled commercial anti-human Ig (Immu
Noglobulin) antibodies can be used.

The sample for immunoassay can be prepared by a conventional method.
For example, a blotting method in which a filter membrane adsorbs nucleic acids and proteins of colonies, plaques or infected cells,
It can be prepared according to a method using a microplate or a slide glass for microscopy. When blotting is used in conjunction with indirect enzyme antibody technology, large numbers of colonies, desired colonies from plaques or infected cells,
Sorting of plaques or infected cells can be performed easily and quickly. In this method, a commercially available filter made of nitrocellulose, cellulose acetate, nylon, or the like is brought into close contact with these colonies, plaques, or infected cells, and blotting is performed.

The cDNA clone thus obtained is
Since it is part of the ANBV gene cDNA, the NANB
In order to obtain a cDNA clone covering the entire region of the V gene, cDNA was isolated by a method in which a cDNA fragment near the cDNA clone was isolated using the 5 ′ or 3 ′ end of the cDNA clone as a probe. It needs to be extended. In this case, Gene Walking (also known as genomic walking or chromo
mouse walking) (“DNA Cloning Volume II”).
I ", edited by DM Glover, 37-39, IRL
Press, 1987; Maniatis et
al. , “Molecular Cloning −
a laboratory manual " 2 , 3.2
1-3.23, 1989). By repeatedly performing the cloning operation and the gene walking, NANBV
The entire gene region can be obtained in the form of a cDNA clone.

At this stage, it is desirable to determine the nucleotide sequence of each obtained cDNA clone. Such a nucleotide sequence of the cDNA clone is determined by a conventional method, for example, the Maxam-Gilbert method, the dideoxy chain terminator method (Analytical Biochemistrr).
y, 152 , 232-238, 1986).

Further, an amino acid sequence can be determined based on the determined base sequence. Sequencing of the amino acids is determined by the initiation codon (ATG or mRN of cDNA).
AUG is sequentially performed by confirming the position of AUG on A. Important portions of such an amino acid sequence, for example, hydrophilic sites considered to constitute an epitope, synthesize peptides corresponding to the respective hydrophilic sites, and purify them by high performance liquid chromatography (HPLC). It can be confirmed by subjecting it to an enzyme immunoassay (EIA) or a radioimmunoassay (RIA).

It is desirable to classify the cDNA clones into several groups in order to distinguish the clones from each other. For example, the position of each cDNA clone on the restriction map of the NANBV gene can be used as an index for classification (FIG. 1-1).
And FIG. 1-2). In addition, since NANBV is known to have a tubular structure forming ability in chimpanzee liver cytoplasm and to have no tubular structure (Science, 205 , 1).
97-200, 1979), serological reaction between the expression product of each cDNA clone, chimpanzee serum infected with NANBV that forms a tubular structure in chimpanzee liver cytoplasm, and chimpanzee serum that does not form a tubular structure. By examining sex, these clones can be identified and classified. Testing for such serological reactivity is
It can be performed by the immunoassay described above.

In the present invention, the prefix “BK” is added to the cDNA clones of the NANBV gene of the present invention shown in FIGS. 1-1 and 1-2. FIG. 1-1 shows the NANB.
It is a figure which shows the position range which each cDNA clone of this invention NANBV gene occupies in the whole region of V gene. FIG. 1-2 is a diagram showing the position range occupied by each cDNA clone obtained by gene walking in the entire region of the NANBV gene.

The NANBV cDNA clone is, for example,
Escherichia coli BK108 (Japanese Patent No. 2971), BK129 (Japanese Patent No. 297)
No. 2), BK138 (Microtechnical Laboratory No. 2973) BK1
53 (Microtechnical Co., Ltd. No. 2974), BK157 (Microtechnical Co., Ltd. No. 3243), BK166 (Microtechnical Co., Ltd. No. 297)
No. 5) and BK172 (Microtechnical Laboratory No. 2976). These seven BK NANBVcD
The NA clone contains at least the entire open reading frame region of the NANBV gene, or
It is considered that the whole region of the ANBV gene is covered.
Furthermore, in addition to the above clones, the following 5 clones have been deposited with the Japan Institute of Fine Arts as representatives of the BK series: BK1
02 (Piercing No. 3384), BK106 (Piercing No. 3385), BK112 (Piercing No. 338)
No. 6), BK146 (Microtechnical Laboratory No. 3387), BK
147 (Microfabrication Kenzo No. 3388).

The nucleotide sequence of the entire region of the NANBV gene covered by the above-mentioned BK NANBV cDNA clone and the amino acid sequence encoded by them are shown in FIGS. 2-1 to 2-16. Based on the entire NANBV nucleotide sequence and the entire NANBV amino acid sequence disclosed in FIGS. 2-1 to 2-16, homology between the nucleotide sequence and the amino acid sequence of the NANBV gene and those of other virus genes, and the hydrophobicity of the amino acid sequence Various studies and observations can be made on the sex index (hydrophobic / hydrophilic profile), the structure of the NANBV gene, the epitope (antigenic determinant) region, and the like.

Regarding homology, the nucleotide sequence and amino acid sequence of the NANBV gene and various known viral genes (Japanese Patent Laid-Open No. 62-286930; Virolog)
y, Volume 161, pages 497-510, 1987
For example, bovine viral diarrhea virus (Viro
165, 497-510, 198
8 years), swine fever virus (Virology, No. 17)
1, 555-567 (1989), tobacco leaf vein mottle virus (Nucleic Acid Rese).
arch, vol. 165, pp. 5417-5430, 1
986).

For the analysis of the hydrophobicity index, for example, SDC-Genet of Genetic Information Processing Software
yx [manufactured by SDC Software Inc. (Japan)], Do
The program created by ottle (Journal
of Molecular Biology, No. 157
Vol., Pp. 105-132, 1982).

FIG. 3 is a diagram showing the hydrophobicity profiles of NANBV of the present invention and Japanese encephalitis virus (JEV), in which the respective hydrophobicity indices of both viruses are compared with each other. From this figure, it has been found that there is significant similarity between the gene structure of the NANBV gene and the gene structure of the JEV gene. As shown in FIG. 3, the polypeptide of NANBV of the present invention is composed of three types of structural proteins, namely, a core protein (C), a matrix protein (M) generated by processing a prematrix protein (PreM), and an envelope protein (E). And seven nonstructural proteins NS1, N
Contains S2a, NS2b, NS3, NS4a, NS4b and NS5. Each of these proteins is encoded by the following base sequence:

The C protein has nucleotide numbers from 333 to 677.
No. 678 to No. 905 for M protein, No. 906 to No. 1499 for E protein, NS1 No. 1500 to No. 2519, NS2 No. 2520 to No. 3350
No. NS3 is from No. 3351 to No. 5177, NS4a
Is 5178th to 5918th, NS4b is 591th
9th to 6371th and NS5 from 6372th to 9362th. These nucleotide sequences are useful for diagnosis of NANB hepatitis, and the polypeptides encoded thereby are useful not only as vaccines for NANBV hepatitis but also as antigens for diagnostic agents.

The above three types of structural proteins are represented by amino acid Nos. 1 (Met) to 389 shown in FIGS. 2-1 to 2-3, and have the first methionine residue. The group is that encoded by the start codon.

Our constant studies have shown that the following nucleotide sequences encode epitopes reactive with anti-NANBV antibodies: base sequences 333 to 422, respectively. No. 333 to 14
No. 99, No. 333 to No. 6371, No. 474 to No. 563, No. 906 to No. 953, No. 1020 to No. 1046, No. 1020 to No. 1121, No. 11
94th to 1232th, 1209th to 1322th
No. 4485 to No. 4574 and No. 5544 to No. 5633.

As described below, the above-mentioned nucleotide sequence or a nucleotide sequence containing a nucleotide sequence as a part of the entire sequence is obtained by NANB by recombinant DNA technology or chemical synthesis.
Not only production of V antigen polypeptide, but also hybridization or polymerase chain reaction (P
CR) can be used effectively for the diagnosis of NANB hepatitis.

The first base sequence containing at least six bases in the entire region of base numbers 1 to 9416 shown in FIGS. 2-1 to 2-16 is used for diagnosis of NANB hepatitis. Useful as a probe for hybridization or as a primer for polymerase chain reaction, wherein at least four amino acids encoded by the nucleotide sequence of at least 12 nucleotides in the nucleotide sequence of nucleotide numbers from 333 to 9362 are used. The contained polypeptide was found to be effective not only as a vaccine for NANB hepatitis but also as an antigen for a diagnostic agent. Furthermore, as is known to those skilled in the art, a second nucleotide sequence complementary to the first nucleotide sequence is also known as NAN
It was found to be useful as a probe for hybridization in diagnosis of hepatitis B or as a primer for polymerase chain reaction. A nucleotide sequence obtained by substituting at least one base of at least a part of the coding region of the first nucleotide sequence of NANBV according to the degeneracy of the genetic codon also includes
It can be used for the production of the antigen polypeptide of the present invention by recombinant DNA technology.

Therefore, the isolated deoxyribonucleic acid of the present invention has the entire nucleotide sequence of the non-A non-B hepatitis virus of base numbers 1 to 9416 shown in FIGS. 2-1 to 2-16. At least one base sequence selected from the group consisting of a first base sequence containing at least a part and a second base sequence complementary to the first base sequence, or the first base sequence according to the degeneracy of the gene codon It includes a base sequence obtained by substituting at least one base.

More specifically, in the deoxyribonucleic acid of the present invention, the first base sequence is shown in FIG.
6 includes at least 6 bases of the entire base sequence of the non-A non-B hepatitis virus of base numbers 1 to 9416 shown in No. 6.

More specifically, in the deoxyribonucleic acid of the present invention, the first base sequence has base number 333.
From No. 422 to No. 333 to No. 677, No. 33
No. 3 to No. 1499, No. 333 to No. 6371,
No. 474 to No. 563, No. 678 to No. 905
No., No. 906 to No. 953, No. 906 to No. 14
No. 99, No. 1020 to No. 1046, No. 1020 to No. 1121, No. 1194 to No. 1232, No. 1209 to No. 1322, No. 1500 to No. 25
No. 19, No. 2520 to No. 3350, No. 3351 to No. 5177, No. 4485 to No. 4574, No. 5178 to No. 5918, No. 5544 to No. 56
It contains at least one base sequence selected from the group consisting of No. 33, No. 5919 to No. 6371, No. 6372 to No. 9362, and No. 1 to No. 9416.

In addition, the isolated antigenic polypeptide of the present invention can be obtained from the nucleotide sequence Nos. 333 to 936 of the nucleotide sequence of the non-A non-B hepatitis virus shown in FIGS. 2-1 to 2-16.
It includes at least a part of the amino acid sequence encoded by the deoxyribonucleic acid containing the second coding region. Note that the NANBV antigen polypeptide of the present invention comprises N
A polypeptide which does not constitute an assembly such as an ANBV particle or a NANBV surface antigen assembly.

More specifically, the antigen polypeptide of the present invention comprises at least one of at least four amino acids encoded by a nucleotide sequence of at least 12 nucleotides in the nucleotide sequence of base numbers 333 to 9362. Amino acids.

More specifically, the antigenic polypeptides of the present invention may have base numbers from 333 to 422,
No. 33 to No. 677, No. 333 to No. 1499,
No. 333 to No. 6371, No. 474 to No. 563
No., No. 678 to No. 905, No. 906 to No. 95
No. 3, No. 906 to No. 1499, No. 1020 to No. 1046, No. 1020 to No. 1121, No. 11
94th to 1232th, 1209th to 1322th
No. 1,500 to No. 2519, No. 2520 to No. 3350, No. 3351 to No. 5177, No. 44
No. 85 to No. 4574, No. 5178 to No. 5918
No. 5544 to No. 5633, No. 5919 to No. 6371, No. 6372 to No. 9362, and Amino acid encoded by a base sequence selected from the group consisting of No. 333 to No. 9362 Contains the sequence.

Further, since the polypeptide encoded by the entire coding region of NANBV shown in FIGS. 2-1 to 2-16 has a broad antigen-antibody reaction spectrum, such a polypeptide may have a single epitope Can react with a wide variety of antibodies produced by NANB hepatitis infection as compared to antigens containing the same, and is highly sensitive as a diagnostic agent for NANB hepatitis.

(VI) Expression of NANBV gene cDNA clone and mass production of NANBV antigen: NANB
NANBV to produce V antigen polypeptides on a commercial scale
To express the cloned cDNA of the antigen gene,
Part or all of the clone cDNA present in the DNA clone is removed from the cloning vector and ligated to the expression vector. For example, a DNA fragment containing the NANBV antigen gene (hereinafter referred to as “NANBV DNA fragment”) is obtained by cutting out a part or the whole region of the cDNA of each cDNA clone using a restriction enzyme, and expressing the DNA fragment by a conventional method. Insert into vector. One gene D
When the NA fragment is inserted into an expression vector, one kind of peptide encoded by the DNA fragment can be produced. When two or more gene DNA fragments are inserted into the sequence of an expression vector, the polypeptide encoded by the inserted DNA fragment can be produced as a fusion peptide by gene expression.

Known or commercially available replicable expression vectors that can be used in this procedure include, for example, enterobacteriaceae plasmid vector pSN508 (US Pat. No. 4,703,005) and yeast plasmid vector p.
BH103 and its series (JP-A-63-22098)
And pJM105 (JP-A-62-286930), attenuated varicella virus gene (JP-A-53-41202), attenuated Marek's disease virus (Japanese Veterinary Society, Vol. 27, 20-
24, 1974; and Gan Monogr
aph on Cancer Research, No. 1
0, pages 91-107, 1971), pTTQ series (manufactured and sold by Amersham (UK)), pSLV series (manufactured and sold by Pharmacia LKB (Sweden))
Etc. can be used.

Next, the expression vector into which the DNA of NANBV has been inserted is individually transferred or transfected into host cells sensitive to the vector by conventional means.
Transformants are obtained, and transformants producing NANBV antigen polypeptide or NANBV particles are selected therefrom. The production of the NANBV antigen polypeptide can be detected by the immunoassay described in (V) above. As a result, when an animal virus gene is used as an expression vector, a recombinant virus producing a NANBV antigen polypeptide can be obtained. Such a recombinant virus has not only an antigenic property inherent in the virus vector but also a NANBV antigen polypeptide. It can be used as a raw material for a multifunctional vaccine that also has antigenicity.

The NANBV antigen polypeptide can be produced on a commercial scale in the roast by culturing the transformant or recombinant virus obtained above in a conventional manner. For details of the method of using an animal virus gene as an expression vector, see EP-A-3345.
You should refer to No. 30.

Further, the present invention provides a non-A
It is intended to provide a method for producing a non-hepatitis B virus antigen polypeptide: (a) after introducing deoxyribonucleic acid into a replicable expression vector selected from a plasmid and an animal virus gene, In the case of a plasmid, a replicable recombinant DNA containing the plasmid and the deoxyribonucleic acid inserted therein is obtained. When the replicable expression vector is an animal virus gene, the animal virus gene and the deoxyribonucleic acid inserted therein are obtained. And the deoxyribonucleic acid is obtained as shown in FIG.
6. Includes at least a partial region of the nucleotide sequence Nos. 1 to 1499 or at least a partial region of the nucleotide sequence Nos. 1500 to 9416 of the nucleotide sequence of the non-A non-B hepatitis virus represented by 6. (B) a base sequence selected from the group consisting of a first base sequence and at least one base sequence obtained by substituting at least one base of the first base sequence by degeneracy of a gene codon; When the replicable expression vector used in (a) above is a plasmid, the recombinant DNA is transfected into a microorganism or eukaryotic cell culture to form a transformant, and then the microorganism or eukaryotic cell culture is cultured. (C) culturing the transformant obtained in the above (b) to express the deoxyribonucleic acid, and culturing the transformant obtained in (b) above. The deoxyribonucleic acid and the animal virus gene are expressed by producing a virus antigen peptide or culturing the recombinant virus obtained in the above (a), and the animal virus gene and the non-A non-B hepatitis virus antigen peptide are expressed. Non-A non-B in the form of recombinant virus produced
Producing a hepatitis B virus antigen peptide, and (d) isolating such a non-A non-B hepatitis virus antigen peptide alone or in the form of a propagating recombinant virus.

The antigen peptide obtained in this way is NAN
It can be used as a diagnostic agent for hepatitis B or as an active ingredient of a vaccine, and a recombinant virus is also used as an active ingredient of a vaccine.

Further, the cDNs shown in FIGS.
Using a part or the whole of A as a template, i
The corresponding RNA or mR transcribed in vitro
NA can be synthesized. For example, RNA or m corresponding to the entire region of the cDNA of FIGS.
RNA was prepared by using the entire region of the cDNA as a template,
Transcribe in vitro using RNA polymerase. RNA or mRNA thus synthesized
Contains the entire region of the NANBV gene, ie
By transfecting mRNA into animal cells, the NANBV genome is substantially naked,
V antigen or infectious NANBV particles can be obtained. In addition, for the synthesis of such mRNA, for example, commercially available mR
An NA capping kit (manufactured and sold by Stratagene (USA)) can be used. For details of the operation, refer to “Current Protocols in
Molecular Biology "(10.1
7.1-10.17.5, issued by Wiley & Sons, 1989). Since the RNA obtained by using a part or the whole of the cDNA of FIG. 2-1 to FIG. 2-16 is a part or the whole of the NANBV genome, not only the production of the NANBV antigen but also the NANBV
And is useful for studying infectious diseases caused by it.

(VII) Purification of NANBV antigen polypeptide: The NANBV antigen produced in the culture of the transformant or the recombinant virus can be obtained by a conventional technique, for example,
Salting out, adsorption / desorption method using silica gel, activated carbon, etc., precipitation method using organic solvent, ultracentrifugation fractionation method, ion exchange or affinity column chromatography separation method, high performance liquid chromatography, electrophoresis fractionation method, etc. Can be purified by appropriately combining them. When purifying the NANBV antigen peptide from a culture of a transformant of E. coli or yeast, NA
In view of the effective removal of allergens from Escherichia coli and yeast, which significantly reduce the quality of the final product of the NBV antigen polypeptide, for example, (1) silica gel adsorption / desorption treatment, activated carbon adsorption / desorption method, and (2) Purification by sequentially performing fractionation by density gradient centrifugation (JP-A-63-297) is desirable. When purifying the NANBV antigen polypeptide from a recombinant virus culture, for example, a virus-infected recombinant cell culture, the antigen can be purified by repeatedly performing ultracentrifugation or density gradient centrifugation. Highly pure NANBV antigen polypeptide can be obtained.

Thus, the purified NANBV of the present invention
A stock solution containing the antigen polypeptide is obtained. If necessary, the solution can be lyophilized to obtain such a NANBV antigen polypeptide in dried form. Further, the mixed antigen polypeptide of the present invention can be obtained by mixing at least two types of NANBV antigen polypeptides obtained by gene expression of at least two types of cDNA having different base sequences.

(VIII) Synthesis of NANB antigen: As described above, the core protein (C protein), matrix protein (M protein), and envelope protein (E protein) of NANBV are shown in FIGS. 2-1 to 2-3. In the region from amino acid No. 1 (Met) to No. 389 (Gly). The above-mentioned epitopes contained in this region, particularly the epitopes encoded by the nucleotide sequences of base numbers 906 to 953, 1020 to 1046, and 1194 to 1232 are very useful as antigens It is. Such an epitope can be obtained by polypeptide synthesis, and a commercially available peptide synthesizer, for example, a polypeptide synthesizer COUPLER 2100 [D
U PONT (USA) sales], Peptide Synthesizer 430
A [APPlied Biosystem (USA)
And the like. Such synthetic antigenic polypeptides can be used, for example, in the production of vaccines, diagnostics, and antibodies.

(IX) Important regions of NANBV cDNA:
The present invention further comprises a replicable expression vector selected from a plasmid and an animal virus gene, and a deoxyribonucleic acid, wherein the deoxyribonucleic acid is shown in FIG.
A first nucleotide sequence containing at least a partial region of nucleotide numbers 1 to 1499 or at least a partial region of nucleotide numbers 1500 to 9416 of the entire nucleotide sequence of the non-A non-B hepatitis virus shown in 16 And a replicable recombination comprising a base sequence selected from the group consisting of at least one base sequence obtained by substituting at least one base for the first base sequence based on the degeneracy of a gene codon. The purpose is to provide the body. Such a recombinant can produce not only the NANBV antigen polypeptide of the present invention but also the NANBV gene cDNA of the present invention by replication.
Can be used for amplification.

The preferred first nucleotide sequence to be inserted into and linked to the above-mentioned recombinant for amplifying the NANBV gene cDNA of the present invention is at least six nucleotides of the nucleotide sequence of nucleotide numbers 1 to 1499. It contains a base or at least 6 bases from the 1500th to 9416th base sequence. In addition, a preferred first base sequence inserted and linked to the above-mentioned recombinant for production of the NANBV antigen polypeptide of the present invention is at least 12 bases of the base sequence of base numbers 333 to 1499, or It contains at least 12 bases from the 1500th to 9362nd base sequences.

Further, the first nucleotide sequence inserted and linked to the above-mentioned recombinant for the production of the NANBV antigen polypeptide of the present invention is as follows: nucleotide numbers 333 to 422; No. 333 to 1499
No., No. 474 to No. 563, No. 678 to No. 90
5th, 906th to 953th, 906th to 1st
No. 499, No. 1020 to No. 1046, No. 1020
No. 1121 from No. 1194 to No. 1232,
No. 1209 to No. 1322, No. 1500 to No. 2
No. 519, No. 2520 to No. 3350, No. 3351
From No. 5177 to No. 4485 to No. 4574,
5178th to 5918th, 5544th to 5th
No. 633, No. 5919 to No. 6371, and No. 6
It is selected from the group consisting of the 372nd to 9362nd base sequences.

(X) Production of diagnostic antigen: The purified NANBV antigen polypeptide of the present invention is useful as a diagnostic agent for hepatitis NANB. The NANBV antigen polypeptide of the present invention can be prepared as a diagnostic agent as follows. The purified NANBV antigen polypeptide solution obtained in the above (VII) is dispensed into a container such as a vial or an ampule, and then sealed. The injected antigen polypeptide can be lyophilized by the same method as described above before sealing, and the amount of the NANBV antigen polypeptide in the container is:
Generally, from about 1 μg to about 10 mg. Instead, the NA
The NBV antigen polypeptide can also be prepared by using a commonly used support such as a microplate, polystyrene beads,
It can be adsorbed on the surface of filter paper or membrane.

The determination of the reactivity between the serum and the NANBV antigen polypeptide can be performed individually by the same means as described in (V) above. That is, the determination of such reactivity is based on conventional immunoassay methods, for example, radi
ommunoassay (RIA), enzyme-
linked immunosorbent assa
y (ELISA), fluorescent antibody method (FA), passive red blood cell agitation reaction (PHA), reverse passive red blood cell agitation reaction (rPHA)
And so on. The amount of the NANBV antigen polypeptide used in the above immunoassay is generally about 0.1 to about 100 mg / ml in serum, and the amount used for RIA, ELISA, FA, PHA, and rPHA is 0.1 to about 100 mg / ml, respectively. 1 mg / ml, 0.1-
1 mg / ml, 1-100 mg / ml, 1-50 mg /
ml and 1 to 50 mg / ml.

The NANBV antigen polypeptide of the present invention can also be used for screening blood for transfusion. Such a screening method comprises: (a) separating serum from whole blood; (b) serum from unknown blood and base numbers 333 to 9 of the entire nucleotide sequence of NANBV shown in FIGS. 2-1 to 2-16.
An isolated NAN containing at least one amino acid comprising at least a portion of the amino acid sequence encoded by a deoxyribonucleic acid comprising the coding region at position 362
(C) determining the presence or absence of a reaction between the serum and the NANBV antigen polypeptide; and (d) determining whether it is positive or negative for non-A non-B hepatitis based on its reactivity. And (e) sorting blood for transfusion based on the result.

The contact of the serum of an unknown blood with the NANBV antigen polypeptide of the present invention and the determination of its reactivity are determined by the NA
It can be carried out by the same means as described above for the method for diagnosing hepatitis NB. By this method, blood for transfusion not infected with NANBV can be selected.

(XI) Production of Vaccine: The NANBV antigen polypeptide of the present invention can be used as an active ingredient of a NANB hepatitis vaccine. Such N
The hepatitis ANB vaccine can be prepared as follows. Culture of a transformant containing a recombinant phage or a plasmid that is a carrier of the cDNA encoding the NANBV antigen polypeptide, and culturing cells infected with the recombinant virus that is a carrier of the cDNA, The NANBV antigen polypeptide is produced. still,
In order to detoxify the NANBV antigen in such a culture and ensure safety, and to immobilize and stabilize the immunogenicity or antigenicity of the antigen, the culture before the purification of the above (VII), Alternatively, it is desirable to inactivate at the stage of culture filtrate or centrifugal supernatant after filtration or centrifugation to remove cell debris or cultured cells from the culture. For example, a common inactivating agent, formalin, is added to a final concentration of 0.0001 to 0.001% (V
/ V) after adding and mixing the above culture preparation so that
By storing at 37 ° C for 5 to 90 days, NANB
V antigen can be inactivated. A vaccine stock solution can be prepared in the same manner as in the above (VII).

Further, the NANBV vaccine stock solution was filtered by a conventional method using a microfilter, and the solution was sterilized.
The filtrate is diluted with a physiological salt solution so that the measured value of the protein concentration by the Lowry method becomes about 1 to about 500 μg / ml,
Add aluminum hydroxide gel as adjuvant to a final concentration of about 0.1 to about 1 mg / ml. Further, at least one stabilizer can be added to such a mixture, and as the stabilizer, a commercially available known stabilizer can be used. Examples of the stabilizer include gelatin, a hydrolyzate thereof, albumin, and glucose glucose. , Fructose,
Galactose, sucrose and lactose, as well as the amino acids glycine, alanine, lysine, arginine, glutamine and the like can be used. Examples of the adjuvant include a precipitated calcium phosphate gel, an aluminum phosphate gel, aluminum sulfate, alumina and bentonite, a muramyl peptide derivative of an adjuvant that induces antigen production, a polynucleotide, Kres.
tin (registered trademark, manufactured and sold by Kureha Chemical Industry Co., Ltd. (Japan)) and Picibanil (both antitumor agents) can be used. Furthermore, at least one stabilizer can be added to such a mixture, and commercially available and known stabilizers include, for example, gelatin, their hydrolysates, albumin, sugar glucose, fructose, galactose, and the like. Sucrose and lactose, as well as the amino acids glycine, alanine, lysine, arginine and glutamine can be used.

The precipitated NANB hepatitis vaccine is obtained by dispensing the NANB hepatitis vaccine solution containing the gel-adsorbed NANBV antigen polypeptide into a small container such as an ampoule or a vial and sealing it.

By lyophilizing the obtained NANB hepatitis vaccine solution to obtain a dried NANB hepatitis vaccine, the preparation can be transported and stored in a harsh environment, for example, a tropical environment. Such lyophilization generally involves liquid settling NAN.
The hepatitis B vaccine is dispensed into containers such as vials and ampoules, followed by a conventional method. After freeze-drying, nitrogen gas is injected into the container containing the dried vaccine and sealed. still,
Regarding the quality of vaccine preparations, the Ministry of Health and Welfare Notification No. 1
The test is carried out in accordance with "precipitated hepatitis B vaccine", "dried Japanese encephalitis vaccine" and "precipitated pertussis vaccine" prescribed in No.59 "Biological Standards".

The NANB hepatitis vaccine comprises the above-mentioned adsorbed NANBV antigen polypeptide and the present NANBV antigen polypeptide.
It can be prepared in the form of a combination vaccine containing at least one antigen other than the antigen polypeptide. Book NAN
Antigens other than the BV antigen polypeptide include the NANB
Side effects and adverse reactions resulting from the combined use of V antigen polypeptides and other antigens are not additively or synergistically increased, and their antigenicity and immunogenicity are reduced by their mutual interference Unless otherwise, it is possible to use any antigen conventionally used as an active factor of the corresponding vaccine. The number and type of antigens that can be mixed with the NANBV antigen polypeptides is such that their side effects and adverse reactions do not increase additively or synergistically, as described above, and their antigenicity and immunogenicity It is not limited as long as it does not decrease. In general, it is possible to mix 2-6 antigens with the NANBV antigen polypeptide, eg, Encephalitis virus, HFRS (nephropathy hemorrhagic fever) virus, influenza virus, parainfluenza virus, B
Hepatitis virus, dengue virus, AIDS virus,
Includes inactivated antigens or detoxified toxins or toxoids from B. pertussis, diphtheria, tetanus, meningococcus, pneumococci, and the like.

In general, a vaccine containing the NANBV antigen polypeptide of the present invention can be provided in the form of a liquid or suspension after being injected into a vial such as an ampule and sealed. When a dried vaccine is used, it is dissolved or suspended in sterilized distilled water to the original volume before freeze-drying before use. Generally, this vaccine is used in a subcutaneous inoculation, the volume of which is given to adults about 0.5 ml, while children receive half the adult vaccine. Such vaccines are usually inoculated twice at an interval of about one week to one month, and one additional inoculation about half a year later.

(XII) Preparation of diagnostic antibody: NANB
V antigen polypeptides can also be used for the production of antibodies such as polyclonal and monoclonal antibodies specific to it. For example, a polyclonal antibody specific to the NANBV antigen polypeptide can be prepared by the following conventional method. First, the purified NA of the present invention
Animals such as mice, guinea pigs and rabbits are inoculated subcutaneously, intramuscularly, intraperitoneally or intravenously several times with an NBV antigen polypeptide at intervals of usually 1 to 4 weeks, and the animals are completely immunized. Conventional commercial adjuvants can be used to enhance the immune effect. Next, after collecting serum from the immunized animal, anti-N
An ANBV antigen polypeptide polyclonal antibody is isolated and purified.

On the other hand, the monoclonal antibody specific to NANBV can be prepared by a conventional method described, for example, in "Cell Engineering", Vol. 1, pp. 23-29 (1982). For example, spleen cells obtained from a mouse immunized with a purified NANBV antigen and commercially available mouse myeloma cells are fused by a cell fusion technique to produce hybridomas, and then these hybridomas are used to prepare the NAN.
Hybridomas that have the ability to produce antibodies reactive with the BV antigen polypeptide are selected and further cloned. Such a clone is cultured by a conventional method, and an anti-NANBV antigen polypeptide monoclonal antibody is isolated and purified from the culture supernatant.

The above-mentioned polyclonal and monoclonal antibodies can also be used as a diagnostic agent for NANB hepatitis. NANB using such an antibody
Diagnosis of hepatitis B can be carried out substantially in the same manner as the above-mentioned method for diagnosis of NANB hepatitis using the NANBV antigen polypeptide. By using a polyclonal antibody or a monoclonal antibody, it becomes possible to identify and quantify the NANBV antigen polypeptide present in liver tissue and blood. Further, the above-mentioned polyclonal antibody and monoclonal antibody are
It can be used as an ANBV remover. For example, the affinity chromatography technique using such an antibody can effectively remove NANBV in blood.

(XIII) Production of Gene Diagnosis Agent: The NANBV gene cDNA of the present invention can be prepared by digesting the NANBV gene cDNA of the present invention with an appropriate restriction enzyme. In addition, the gene cDNA of the present invention
Can be prepared by a DNA synthesis technique according to the nucleotide sequences shown in FIGS. 2-1 to 2-16 of the present specification.
Preparation of the NANBV gene cDNA by DNA synthesis is performed by using a DNA synthesizer model 380B [Applied B
iosystem (USA), DNA Synt
It can be carried out using a known DNA synthesizer such as a Hesizer model 8700 (manufactured and sold by Biosearch (USA)). NANBV gene cD of the present invention
In genetic diagnosis of NANBV infection, NA can be used, for example, as a primer for polymerase chain reaction (PCR) to detect the NANBV gene in a patient's body fluid or cells. For the diagnosis by polymerase chain reaction, the NANBV gene cD
NA of 10 to 100 ng is used.

The NANBV gene cDNA of the present invention
Can also be used for diagnosis of NANB hepatitis by hybridization technology. That is, the NANBV
The gene cDNA can be labeled with, for example, biotin, alkaline phosphatase, radioisotope ³² P, or the like, and used as a probe for diagnosis by hybridization technology. In this case, first, the recombinant phage containing the NANBV cDNA obtained in the above (V) is treated with an appropriate restriction enzyme to obtain a DNA containing the NANBV cDNA.
After cutting out the fragment, it is ligated to a commercially available replicable cloning vector to obtain a recombinant plasmid containing this DNA fragment. The recombinant plasmid is introduced into a host cell to form a transformant, which is cultured to grow the recombinant plasmid.
A is simply extracted, digested with a restriction enzyme, and subjected to low melting point agarose gel electrophoresis to isolate and purify cDNA encoding the NANBV antigen polypeptide. Thus the cDNA obtained, biotin, alkaline phosphatase and radiolabeled ^{32 P} or the like. In addition, a commercially available nick translation kit or a multi-prime DNA labeling system [Amersh]
am (UK), Nippon Gene (Japan), etc.]. The labeled cDNA is about 5-2
Place in a 0 ml container such as ampoule and seal. This cD
The NA amount is 1 to 100 ug per container, and can be provided in a liquid or lyophilized state. Diagnosis of NANB hepatitis using the labeled cDNA can be performed by a known standard hybridization method. That is, plasma, serum or leukocytes obtained from a patient are brought into contact with a labeled cDNA, and a nucleic acid hybridized with the cDNA is detected by a conventional method. When such cDNA is labeled with an enzyme, detection is performed by enzyme immunoassay, and when the cDNA is labeled with a radioisotope, detection is performed by, for example, scintillation measurement.

[0073]

The present invention will be described below with reference to examples. However, the present invention is not limited only to the following examples. Example 1 (1) Preparation of Plasma-Derived RNA to Prepare cDNA Complementary to NANBV Genomic RNA: To obtain NANBV from plasma, blood glutamic-pyruvic transaminase (GPT) value (Wrob)
Lewisiki, F & J. S. LaDue: Ser
um glutamic pyruvic trans
aminosein cardiac and hep
atic disease. Proc. Soc. Ex
p. Biol. Med. , 91: 569, 1956).
However, a human plasma of 4.81 showing 35 IU / l or more was overlaid on a sucrose 30% (W / W) solution, and then centrifuged (48,000 ×
g, 13 hours, 4 ° C.), and the precipitate was washed with 50 mM Tris.
Suspended in HCl pH 8.0-1 mM EDTA solution and centrifuged again (250,000 × g, 3 hours, 4 ° C.)
Thereafter, the obtained precipitate was dissolved in 75 ml of a 5.5 M GTC solution (5.5 M guanidine thiocyanate, 20 mM sodium citrate pH 7.0, 0.05% sarcosil (sodium lauryl sarcosine), 0.1 M 2-mercaptoethanol). And 16 ml of CsTFA
After overlaying on a 0.1 M EDTA solution (p = 1.51), the RNA is precipitated by centrifugation (140,000 × g, 20 hours, 15 ° C.). The protein and DNA in the supernatant are removed by suction, and the precipitate is washed with 200 μl of TE (10 mM Tr).
It is dissolved in an is.HCl (pH 8.0, 1 mM EDTA) solution, added with 20 μl of 3M sodium chloride and ethanol, and left still at −70 ° C. for 90 minutes. Centrifugation (12,000
0 × g, 30 minutes, 4 ° C.), dissolve the precipitate in TE, add sodium chloride and ethanol again as above,
After standing at 70 ° C., the resulting precipitate was collected and 10 μl of TE
And purified RNA.

(2) Preparation of Liver-Derived RNA for Preparation of cDNA Complementary to NANBV Genomic RNA: Okayama et al.
kayama, M .; Kawaichi, M .; Brown
Stein, F .; Lee, T .; Yokota, and
K. Arai: High-Efficiency Cl
oning of Full-Length cDN
A; Construction and Screeneni
ng of cDNA Expression Lib
raries for Mammarian Cell
s. Methods in Enzymology 1
54 . 3-28, 1987) NANBV genomic RNA
Was prepared.

That is, 1 g of the resected liver was fragmented, and the above (1)
Was suspended in 100 ml of 5.5 M GTC solution and homogenized using a Teflon-glass homogenizer.
Next, using a syringe with a # 18 injection needle, the homogenate was taken in and out, and the DNA was cut mechanically. The supernatant obtained by low-speed centrifugation (1,500 × g, 15 minutes, 4 ° C.) was subjected to Cs by substantially the same method as described in (1) above.
After overlaying on a TFA solution and centrifuging, the resulting precipitate RNA
Fractions were resuspended in 0.4 ml of 4M GTC solution and
Add 1 μl of 1 M acetic acid and 300 μl of ethanol,
Let stand at −20 ° C. for at least 3 hours to precipitate RNA. After centrifugation (12,000 × g, 10 minutes, 4 ° C.), the precipitate was dissolved in 1 ml of a TE solution, and 100 μl of a 2M sodium chloride solution and 3 ml of ethanol were added.
The mixture was allowed to stand at 0 ° C. for 3 hours, and the resulting precipitate was collected by centrifugation.
The liver-derived RNA was purified by dissolving in 0 μl of TE.

(3) Preparation of Double-Stranded cDNA Using cDNA Synthesis Kit: A double-stranded cDNA was prepared using a commercially available cDNA synthesis kit (available from Amersham, UK). That is,
To 0.75 μg of the purified RNA obtained in the above (1), reagents attached to the kit, 2 μl of a random hexanucleotide primer, and 2 μl of a reverse transcriptase solution, make up a total volume of 20 μl with distilled water, and bring the reaction solution to 42 ° C. For 40 minutes to synthesize a first strand of cDNA, and then, while cooling in ice water, synthesis of a second strand. First, 37.5 μl of a second strand synthesis reaction buffer attached to the kit was added to 20 μl of the reaction solution.
And 1 μl of E. coli ribonuclease H and 6.6 μl of DNA polymerase I, and 34.9 μl of distilled water were further mixed. 60 minutes at 12 ° C, then 60 ° C at 22 ° C.
After incubation for 10 minutes, further incubate at 70 ° C for 10 minutes,
Cool again with ice water and add 1 μl of T4 DNA polymerase.
After incubating at 37 ° C. for 10 minutes, 4 μl of 0.25 M EDTA (pH 8.0) was added to stop the reaction, phenol / chloroform was mixed well, and then centrifuged (12,000 × g, 1 minute). The aqueous layer was separated. The aqueous layer was further subjected to the above-mentioned extraction operation once more, and then an equal volume of chloroform was added. After stirring well, the aqueous layer was separated by centrifugation, and then an equal volume of 4M ammonium acetate and 2 volumes of ethanol were added. The purified double-stranded cDNA was precipitated at -70 ° C. The precipitate was dissolved in 50 μl of 2M ammonium acetate, added with 100 μl of ethanol, precipitated again at −70 ° C., collected by centrifugation (12,000 × g, 10 minutes), dried, and dried, and then 20 μl of TE. Was dissolved.

(4) Preparation of Double-Stranded cDNA by Polymerase Chain Reaction (PCR) Using the RNA prepared in the above (1) and (2) as a template, cDNA prepared using reverse transcriptase was
R method (Saiki, RK; Gelfand, DH;
Stoffer, S; J; Higu
chi, R; Horn, G .; T; Mullis. K ・
B; and Erlich, H .; A. : Primer-
directed enzymatic amplifier
Iteration of DNA with a the
rmostable DNA polymerase.
Science 239 : 487-491, 198
Amplified by 8). That is, RNA 5 to 1,000 ng
With 20 μl of reverse transcriptase solution ｛50 mM Tris · HC
l pH 8.3, 40 mM KCl, 6 mM MgC
l ₂ 1 µM 3'-primer G (oligonucleotide consisting of 25 bases from base number 7949 to base number 7973 in Figure 2-14), 10 mM dNTP and 0.5 units of reverse transcriptase [New England B
io Lab. , (USA)]} for 30 minutes at 37 ° C. Next, 80 μl of the PCR reaction solution {18 mM Tris · HCl pH 8.3, 48 m
M KCl, 1.5 mM MgCl ₂ , 0.6 μM each of 5′-primer (base number 7612 in FIG. 2-13)
No. 7636 to a synthetic oligonucleotide containing 25 bases) and the above 3′-primer, 10 mM
DNTPs and 2.5 units of Taq DNA polymerase [Perkin Elmer Cetus, USA]
Production and sales] at 94 ° C for 1 minute, 50 ° C for 2 minutes, 72
The incubation at 3 ° C. for 3 minutes was repeated 40 times, and the amplified cDNA was recovered by agarose gel electrophoresis. After phenol treatment and ethanol precipitation, it was dried and dissolved in 10 μl of TE.

(5) Preparation of cDNA Library Using Lambda gt11: Commercially Available cDNA Cloning Kit [Amersham
(UK)). That is, 130 ng of the cD prepared in (3) above
L / K buffer 2μ attached to the cloning kit for NA
l, 2 μl of EcoRI adapter and 2 μl of T4 DNA ligase, and further adjust the total volume to 20 μl with distilled water.
After incubation at 5 ° C. for 16-20 hours, 0.25M
The reaction was stopped by adding 2 μl of EDTA. Then
Pass the size fractionation column attached to the kit and
After removing the EcoRI adapter not linked to NA, 83 μl of L / K buffer and 8 μl of T4 polynucleotide kinase are added to 700 μl of the EcoRI adapter-ligated cDNA and incubated at 37 ° C. for 30 minutes. Then, phenol extraction was performed twice, concentrated to 350 to 400 μl with butanol, and ethanol precipitated was dissolved in 5 μl of TE.

Next, the cloning vector lambda gt1
EcoRI adapter-added cDN at EcoRI site 1
To insert A, 1 μl (10 ng) of the above-described EcoRI adapter-ligated cDNA, 1 μl of L / K buffer, 2 μl (1 μg) of lambda gt11 arm DNA and T4
2 μl of DNA ligase was added, and the total volume of the mixture was adjusted to 10 μl with distilled water, followed by incubation at 15 ° C. for 16 to 20 hours to prepare a recombinant gt11 gt11 DNA solution. Further, in order to obtain a recombinant lambda phage by performing in vitro packaging, 10 μl of A solution of Gigapack II Gold of a commercially available in vitro packaging kit [manufactured by Stratagene (USA)].
And B solution (15 μl) were added to the above recombinant lambda gt11 DNA solution (4 μl), and the mixture was incubated at 22 ° C. for 2 hours.

(6) cDNA cloning using Escherichia coli plasmid pUC19: Using a commercially available DNA ligation kit [manufactured by Takara Shuzo Co., Ltd. (Japan)], cDNA was transformed into Escherichia coli plasmid pUC19 (C.Yanishi-P).
erron, J .; Vieira, J. et al. Messing,
Gene 33, 103, 1985) and cloned in E. coli. That is, the cD prepared by the polymerase chain reaction (PCR) method in (4) above
After cleavage with 5 μl of NA and restriction enzyme SmaI, 40 μl of solution A and 10 μl of solution B added to the kit were added to 5 μl (50 ng) of dephosphorylated plasmid pUC19 DNA,
Incubate at ° C for 16 hours. Escherichia coli JM109 strain (Mess
ing, J; and Seeburg,
P. H. , Nucleic Acids Res 9,
309, 1981) using the calcium chloride method (Mande).
1, M .; and A. Higa, J .; Mol. Bio
l. , 53, 154, 1970).
Transformed Escherichia coli containing the plasmid into which the DNA was incorporated was obtained.

(7) NAN from cDNA library
Screening of clones having BV gene: Escherichia coli strain Y1090 (Richard A. Young and
d Ronald W. Davis, Science,
222, 778, 1983) in 37 ml of 50 ml of LBM medium (1% tryptone, 0.5% yeast extract, 1% sodium chloride, 50 μg / ml ampicillin and 0.4% maltose) at a logarithmic growth phase. Were suspended in 15 ml of ice-cold 10 mM magnesium sulfate. The phage solution obtained in the above (5) was added to an SM buffer (0.1 M sodium chloride, 8 mM magnesium sulfate,
50 mM Tris. HCl pH 7.5, 0.01%
Gelatin) and 0.1 ml of this diluted phage solution
And the above bacterial solution were mixed in equal amounts, incubated at 37 ° C. for 15 minutes, and then heated to 45 ° C. in 4 ml of soft agar medium (1).
% Tryptone, 0.5% yeast extract, 0.5% sodium chloride, 0.25% magnesium sulfate and 0.7% agar PH 7.0) and add L-agar medium (1% tryptone, 0.5% yeast) Extract, 1% sodium chloride, 100
μg / ml ampicillin PH 7.0, 1.5% agar)
And incubated at 42 ° C. for 3 hours. Then, 10 mM IPTG (Isopropyl)
β-D-thiogalactopyranosid
e) is impregnated with a nitrocellulose filter, and the dried one is closely adhered to a plate.
For 3 hours. The separated filter was washed three times with TBS buffer, immersed in 2% bovine serum albumin, and incubated at room temperature for 1 hour. 1/20 volume of the attached E. coli lysate was added to a commercially available immunoscreening kit (available from Amersham (UK)), incubated at room temperature for 30 minutes, and then 50 times with 0.2% bovine serum albumin-added TBS buffer. Diluted NA
This filter was immersed in the serum of a patient with hepatitis NB and reacted at room temperature for 1 hour.

The obtained filter is 0.05% Tween
After washing four times with n20-added TBS buffer, 1 part of peroxidase-labeled anti-human IgG [manufactured by Cappel (Germany)] was added.
Immerse in the antibody solution diluted 000-fold for 1 hour.
After washing with n-TBS solution, 0.4 ml of DAB (3,3′-diaminoben) was added to 50 ml of TBS buffer.
zidine tetrahydrochlorid
e) and 15% of a 30% hydrogen peroxide solution.
After incubating for 5 to 30 minutes at room temperature to develop color, the filter was thoroughly washed with distilled water to stop the reaction.

The plaque obtained by the above operation was purified, and nine positive clones were isolated. These clones were designated BK102, BK103, BK105, and B, respectively.
K106, BK108, BK109, BK110, BK
111 and BK112. None of these clones reacted with normal human serum, but specifically reacted with NANB hepatitis patient serum (Table 1).

[0084]

(8) Determination of Nucleotide Sequence of Obtained Clone: Recombinant Phage D of Clones BK102 to BK112
After recovering NA and digesting with restriction enzyme EcoRI, NANB
V cDNA fragments were isolated and individually isolated on plasmid pUC1.
The Escherichia coli JM109 strain was transformed by substantially the same method as in the above (6) using the obtained plasmid. Plasmid DNA was purified from these transformed Escherichia coli and purified by a 7-DEAZA sequence kit [manufactured by Takara Shuzo Co. (Japan), Mizusawa,
S. , Nishimura, S .; and Seela,
F. , Nucleic Acids Res. , 14,
1319, 1986] using the respective NANBVcDN
The nucleotide sequence of A was determined. FIG. 1-1 shows the relationship between the nucleotide sequences of the obtained cDNA clones.

(9) Cloning of NANBV cDNA clone from cDNA library by gene walking: The cDNA fragments of clones BK102, BK106 and BK112 obtained in (8) above were cloned into ³² P-dCT
A probe labeled with P was prepared, and NANBVcD was hybridized from the cDNA library of the cloning vector lambda gt11 obtained in (5) above.
A phage clone containing NA was obtained. That is, clones BK102, BK106 and B obtained in the above (8)
The alkaline method (Mania) was used from the transformed E. coli K112.
tis T. Fritsch E. et al. F. , And S
ambrook J. : Isolation of B
acteriophage and Plasmid
DNA: "Molecular Cloning" Co
ld Spring Harbor Lab. , Pp7
5-96. ) To prepare a plasmid DNA.

The plasmid DNA of clone BK102 was digested with restriction enzymes NcoI and HincII, and the resulting 0.7 Kb fragment at the 5 ′ end of the DNA was recovered after agarose gel electrophoresis. Plasmid DNA of clone BK106 and clone BK112 was obtained using restriction enzyme Nc
digested with oI and clone BK106
From the DNA fragment of 1.1 kb and clone BK
From 312, a 0.7 Kb fragment at the 3 'end was recovered. 25 ng to 1 μg of the DNA fragment was
Using an NA labeling kit (Nippon Gene) [α- ³² P] dCTP [3000 Ci / mmol, A
A probe for hybridization was prepared by incubating at 37 ° C. for 3 to 5 hours.

Next, the phage of the cDNA library obtained in the above (5) was ligated as described in (7) above.
After incubating at 42 ° C. for 3 hours and then at 37 ° C. for 3 hours on an agar medium, the mixture was cooled, and a nitrocellulose filter was placed thereon and left for 30 to 60 seconds to adsorb phage to the filter. Alkaline denaturation with 0.5N sodium hydroxide-1.5M sodium chloride solution for 1-5 minutes, 0.5M
After neutralizing with Tris · HCl pH 8.0-1.5M sodium chloride solution for 1 to 5 minutes, washing with 2 × SSC solution (0.3M sodium chloride-0.03M sodium citrate),
After air drying, baking was performed at 80 ° C. for 2 hours.

The filter was washed with a hybridization solution (50% formamide, 5 × SSC, 5 × Denha).
rt solution, 50 mM phosphate citrate buffer pH 6.5,
100 μg / ml salmon sperm DNA and 0.1% SDS)
After incubating at 42 ° C. for 6 hours at
The plate was immersed again in 300 ml of the above-mentioned hybridization solution to which 1 ml of a probe of × 10 ⁸ cpm / ml was added, and 42
Incubate at 16 ° C for 16-20 hours, 0.1% 2
4 times with SDS solution containing × SSC, 0.1% 0.1 × S
After washing twice with an SDS solution containing SC, drying and autoradiography were performed to isolate clones showing positive hybridization. As a result, clone BK102
Clone, clone BK, which reacts with the probe of origin
14 clones that react with the 106-derived probe, and
Thirteen clones that reacted with the probe derived from clone BK112 were obtained, and each clone was named BK114 to BK169 in order.

Further, the nucleotide sequence of each of clones BK114 to BK169 was determined as described in (8) above,
When each clone was mapped, a nucleotide sequence having a length of about 9.5 Kb, which is considered to be almost the entire length of the NANBV genome, was mapped (FIG. 1-2). Among them, 5 '
From the clone BK157 located on the terminal side, the restriction enzyme Kp
A 0.55 Kb fragment at the 5 ′ end was recovered by nI digestion, and clone BK116 located at the 3 ′ most terminal was
The 0.55 Kb fragment at the 3 'end was recovered from the digestion with the restriction enzymes HpaI and EcoRI, and ³² P
The probe labeled with is prepared, and c obtained in the above (5) is prepared.
As a result of plaque hybridization of the phage of the DNA library, three new clones were separated with a probe derived from clone BK157, and named clones BK170, BK171 and BK172, respectively.

(10) Analysis of the nucleotide sequence of cDNA: NANB was obtained from the nucleotide sequence of the clone obtained by the above (8) and (9).
The entire nucleotide sequence of the V gene was determined, and FIG.
6 is shown. As a result, the cloned NANBV
The gene cDNA consists of 9416 bases, of which 90
An open reading frame consisting of 30 bases was present, and it was presumed that a protein of 3010 amino acid residues was encoded. The hydrophilic / hydrophobic pattern of this protein is based on the previously reported flavivirus pattern (Sumiyoshi H., Mori).
C. , Fuke I .; et al. , Complete
e Nucleotide Sequence of
the Japanese Encephalitis
Virus Genome RNA. Virolog
y, 161, 497-510, 1987). Representative clones include BK157 (base numbers 1 to 1962), clone BK172 (5 to 366), clone BK153 (338 to 1802), and clone BK138 (1755 to 5124). No.), clone BK129 (No. 4104 to No. 6973), clone BK108 (No. 6886 to No. 8344), and clone BK166 (No. 8).
082 to 9416). These clones, respectively, are Escherichia co.
li BK108 (Microtechnical Lab. No. 2971), BK1
29 (Microtechnical Co., Ltd. No. 2972), BK138 (Microtechnical Co., Ltd. No. 2973), BK153 (Microtechnical Co., Ltd. No. 297)
No. 4), BK157 (Microtechnical Laboratory No. 3243), BK
166 (Piercian Laboratories No. 2975) and BK172
(No. 2976, Microtechnical Laboratory). In addition to the above clones, the following 5
Clones have been deposited with MIC: BK102 (Microtechnical Co., Ltd. No. 3384), BK106 (Microtechnical Co., Ltd., No. 33)
No. 85), BK112 (Microtechnical Laboratory No. 3386), B
K146 (Japanese Patent No. 3387) and BK147 (Japanese Patent No. 3388).

(11) NA related to antibody response associated with NANBV infection
Production of NBV-related antigen in E. coli: clones BK106 and BK11 obtained in (8) above
1, and plasmid DNAs into which the cDNAs of BK112 and clone BK147 obtained in (9) above were individually inserted were collected by a conventional alkaline method. Then
The recovered DNA of clone BK106 is a restriction enzyme Ec
digested with oRI and ClaI, 0.34 Kb DNA
A 0.5 μg fragment was obtained, and a T4 DNA polymerase reaction solution (67 mM Tris · HCl pH 8.8, 6.7 mM
Magnesium chloride, 16.6 mM ammonium sulfate, 1
0 mM 2-mercaptoethanol, 6.7 μM EDT
A, 0.02% bovine serum albumin, 0.3 mM dN
(TP and 2-5 units of T4 DNA polymerase) at 37 ° C. for 60 minutes to blunt both ends. Clone BK102 DNA is restriction enzyme Ba
A 0.7 Kb DNA fragment digested with mHI and collected.
5 μg was blunt-ended using T4 DNA polymerase as described above. The DNA of clone BK147 was digested with the restriction enzyme Sau3AI, and the recovered 1 Kb DN
0.5 μg of the A fragment was blunt-ended in the same manner as described above. In addition, the DNA of clone BK111 contains restriction enzyme Ec
0.5 μl of a 1 Kb DNA fragment recovered by digestion with oRI
g was blunt-ended in substantially the same manner as described above. Next, the expression vector pKK233-2 (Amann, E. et al.
and Brosius, J.M. ATG vector f
or regulated high-level e
xpression of cloned genes
in Escherichia coli . Gene,
40, 183, 1985) with the restriction enzyme Hin.
After digestion with dIII, 2 μg of this DNA was added to an S1 nuclease solution (0.3 M sodium chloride, 50 mM sodium acetate pH 4.5, 1 mM zinc sulfate and 100-200
In 1 unit of S1 nuclease) at 37 ° C. for 20 minutes, 0.12M EDTA and 1M T
The reaction was stopped by adding 1/10 volume of ris · HCl (pH 9.0), and phenol extraction was performed. Then, the vector DNA having blunt ends was precipitated and recovered with ethanol. On the other hand, the DNA of the vector pKK233-2 was digested with the restriction enzyme PstI, purified by phenol extraction and ethanol precipitation, and 2 μg of this DNA was used for the above T4DN.
The ends were made blunt by the A polymerase reaction. The D derived from clones BK106 and BK111 thus obtained
0.5 μg of the NA fragment was cleaved with the restriction enzyme HindIII, and 0.5 μg of the DNA fragment derived from clones BK102 and BK147 was cleaved with the restriction enzyme PstI, and mixed with 0.5 μg of blunt-ended vector DNA. 10 × ligation solution (500 mM T
ris · HCl pH 7.5, 100 mM magnesium chloride, 100 mM DTT, 10 mM ATP), 30
Add 0-400 units of T4 DNA ligase and distilled water to make a total volume of 20 μl, incubate at 14 ° C. for 12-18 hours, and place each of the resulting plasmids in pCE
-06, pE-11, pB-02, and pS-09. Next, using these plasmid DNAs, Escherichia coli JM109 strain was transformed substantially as described in (6) above to obtain transformed Escherichia coli, and LB medium (1% W
/ V tryptone, 0.5% W / V yeast extract, and 1% W / V sodium chloride, pH 7.0.
5) The cells were cultured at 37 ° C. in the logarithmic growth phase with 1 mM IPT.
G (isopropyl-β-D-thiogalactopyranoside)
Was added, and the cells were further cultured for 3 hours. Further, E. coli cells were collected by centrifugation (10,000 × g, 15 minutes),
M Tris.HCl (pH 8.0), sonication (20 KHz, 600 W, 5 minutes), and centrifugation (1
(000 × g, 15 minutes) to separate into a supernatant and a precipitate, each of which was sample buffer (20% V / V glycerol, 0.1 M Tris.HCl pH 6.8, 2%
W / V SDS, 2% V / V 2-mercaptoethanol, and 0.02% BPB) and 100 ° C.
For 3 minutes, electrophoresed on 0.1% SDS-7.5% polyacrylamide gel, separated the protein, and
The DNA was transferred to a nitrocellulose filter using a blot cell (available from BIO RAD, USA). Next, the filter was immersed in a 3% gelatin solution, allowed to stand for 60 minutes, and then incubated with a 100-fold diluted serum of a patient with hepatitis NANB at room temperature for 2 to 3 hours. HCl pH 7.5, 0.5M
Sodium chloride and 0.05% V / V Tween
20). Then, the plate was immersed in a 2000-fold diluted peroxidase-labeled anti-human IgG antibody solution,
Incubated for 0 minutes. And distilled water and TT
After washing with BS, color former D as described in (7) above
The reaction was stopped by immersing in a buffer solution containing AB and a 30% hydrogen peroxide solution of a substrate for 5 to 30 minutes, and washing with water.

As a result, as shown in Table 2, the antigens produced by any of the plasmids specifically reacted with the sera of patients with NANB hepatitis, and the cD
The protein produced by NA was shown to be clinically important.

(12) Purification of NANBV-related antigen produced by Escherichia coli and reactivity with sera from patients with liver disease: The usefulness of the proteins produced by cDNA inserted into the expression vector is to purify these proteins by ELISA or It was shown by using it as an antigen for radioimmunoassay. That is, (1)
The cell lysate of the transformed E. coli obtained in 1) is centrifuged (10,
(000 × g, 15 minutes).
For example, the precipitate obtained from the transformant JM109 / pCE066 is subjected to 100 mM Tris · HCl (pH 8.
0) After suspending in -0.1% Triton X-100 solution and sonication (20 kHz, 600 W, 1 minute),
Centrifuge (21,000 xg, 15 minutes) to precipitate
After resuspension in 0 mM Tris.HCl (pH 8.0) -6M urea, sonication was performed.

The obtained supernatant was dialyzed against 10 mM phosphate buffer (pH 7.5) -6M urea, and the antigen solution was diluted with 20 mM urea.
ml, adsorbed to a hydroxyapatite packed column (21.5 x 250 mm) equilibrated with the above buffer by high performance liquid chromatography (HPLC), and eluted with a 0 to 2M sodium chloride linear gradient in the above buffer. The fractions containing the antigen were pooled and dialyzed against 50 mM carbonate buffer (pH 9.6) -0.05% sodium dodecyl sulfate (SDS).

Further, the transformant JM109 / pB-02
Centrifugal supernatant of the cell lysate of -10 (10,000 × g, 1
For 5 minutes) and treated with 35% saturated ammonium sulfate.
It was dissolved in 100 mM 2-mercaptoethanol and dialyzed against the same buffer. Next, the dialyzed sample 100 was applied to a DEAE-cellulose column (22.0 × 200 mm) equilibrated with the same buffer by high performance liquid chromatography.
ml of 50 mM Tris · HCl (pH
8.5) A 0-2M linear gradient elution of sodium chloride was performed in -100 mM 2-mercaptoethanol, and the fractions containing the antigen were pooled.

This fraction was made up of 10 mM phosphate buffer (pH
6.8) After dialyzing against -100 mM 2-mercaptoethanol and adsorbing to a hydroxyapatite column for HPLC equilibrated with the same buffer, 10-40 of phosphoric acid
Elution with a 0 mM linear gradient was performed, and the fractions containing the antigen were pooled, and 50 mM carbonate buffer (pH 9.6) -0.05%
It was dialyzed against SDS solution. Furthermore, the transformant JM109 / p
The centrifuged precipitate of the cell lysate of E-11-89 was suspended in a 10 mM phosphate buffer (pH 5.5), sonicated for 1 minute, and centrifuged (21,000 × g, 15 minutes). The resulting precipitate is 100 mM carbonate buffer (pH 10.5)-
The cells were suspended in a 500 mM sodium chloride-10 mM EDTA solution, sonicated again for 1 minute, and the centrifuged supernatant was added for 30 minutes.
Dialysis was performed against mM phosphate buffer-6M urea. Then
Equilibrated with the same buffer used for dialysis (HPLC)
20cm for CM-cellulose column (22x200mm)
ml of the dialysate was adsorbed and a linear gradient elution of sodium chloride of 0 to 1.5 M was performed in the same buffer. Fractions containing the antigen were pooled, and 50 mM carbonate buffer (pH 9.6)-
It was dialyzed against 0.05% SDS to obtain an antigen solution.

The antigen prepared as described above was used for ELISA.
Was used for clinical diagnosis of NANB hepatitis virus infection. That is, the above purified antigen was prepared at a protein concentration of 1 μg / ml, and ELISA microplate Imron 600 [IMMULON (registered trademark),
100 μl in each well of Greiner (Germany)
Aliquot each and leave at 4 ° C overnight. Each well is PBS
-T buffer (10 mM phosphate buffer pH 7.2, 0.1 mM
8% sodium chloride and 0.05% Tween2
After washing well three times in 0), 100 µl / well of a test serum diluted with a PBS-T buffer was added and reacted at 37 ° C for 1 hour. After washing with PBS-T buffer three times, a peroxidase-labeled anti-human IgG antibody [manufactured by Cappel (Germany)] diluted 8,000-fold with PBS-T buffer containing 10% fetal bovine serum was added to each well. , And reacted at 37 ° C. for 1 hour, washed again 4 times with PBS-T buffer, and treated with a substrate coloring agent solution (9 ml of 0.05M 0.05M).
100 μl of each well containing 0.5 μg of o-phenylenediamine and 20 μl of hydrogen peroxide solution in a citrate-phosphate buffer solution was added to each well, and the plate was allowed to stand at room temperature for 60 minutes while shielding light. 75 μl of 4N sulfuric acid was added to each well, and 4
The absorbance at 90 nm was measured. As is clear from the results shown in Table 3, the antigens derived from all the transformants were NAN.
The specific reactivity with hepatitis B patient serum demonstrated the usefulness of the antigens produced by these transformants in clinical diagnosis.

[0099]

The results shown in Table 3 could also be obtained by radioimmunoassay using the above antigens.
That is, 0.2 ml of the purified antigen solution having a concentration of 1 μg / ml.
Then, a 1/4 inch diameter polystyrene ball (manufactured by Pezel, Germany) is added, and the mixture is allowed to stand at 4 ° C. overnight. Next, the polystyrene balls were washed five times with the PBS-T buffer used in the above ELISA, and the test serum was washed with PBS-T.
200 μl of the solution diluted 20 to 2500 times with the buffer is added thereto, and the mixture is reacted at 37 ° C. for 60 minutes. After washing five times with a PBS-T buffer solution, adding 200 μl of ¹²⁵ I-labeled anti-human IgG antibody and reacting at 37 ° C. for 1 hour,
Washed 5 times with PBS-T buffer and bound to styrene balls. By measuring the ¹²⁵ I cpm, the third
The same results as shown in the table were obtained, indicating the usefulness of the purified antigen obtained previously in clinical diagnosis of NANB hepatitis virus infection.

Application Example 1 Test for Reactivity of Synthetic Polypeptide: Antibody molecules bind to the structure of specific regions known as “epitope” present on antigen molecules, but these specific regions Found in hydrophilic regions. It is considered that an antigen polypeptide having such a specific region can easily prepare a useful clinical diagnostic agent having high reaction specificity. The epitope of NANBV is shown in FIG.
This is estimated from the hydrophilic / hydrophobic pattern of the amino acid sequence encoded by the NANBV gene cDNA shown in -16. That is, base numbers 333 to 422 shown in FIG. 2-1, base numbers 474 to 563 shown in FIGS. 2-1 and 2-2, and base numbers shown in FIG. 2-8. Nos. 4485 to 4574, and FIG. 2-10
Polypeptide B containing the amino acid residues encoded by base numbers 5544 to 5633, respectively
KP-106-1, BKP-106-2, BKP-10
2-1 and BKP-147-1 were synthesized. Each polypeptide was prepared at a concentration of 1 μg / ml and
The solid phase is immobilized on a microplate for ELISA by the same method as described in (12) of
The reactivity with hepatitis B patient serum was tested. As is clear from the results shown in Table 4, all of the synthetic peptides were N
Because it reacts specifically with the serum of hepatitis ANB patients,
The significance of the specific region of the above nucleotide sequence in clinical diagnosis was shown.

[0102] Further, epitopes on the envelope protein of NANBV were estimated, and three polypeptides were synthesized. That is, FIG.
2 and base numbers 906 to 953 and 1020 to 1046 shown in FIG.
The polypeptide encoded by base numbers 1194 to 1232 shown in No. -3 was synthesized. All of these polypeptides correspond to the envelope region in which antigenic mutation is considered to be caused by the NANBV strain, and the reaction with the serum of the NANB hepatitis patient was confirmed by the ELlSA method. The above-mentioned proteins are important and useful as antigens for vaccines and the like in epidemiological investigations and clinical diagnoses.

Application Example 2 PCR (Polymerase Chain Reac)
Detection of NANBV Nucleic Acid by Tion Method: To prevent the onset of NANB hepatitis after blood transfusion, it is important to test for the presence or absence of NANBV infection in blood subjected to blood transfusion. NANBV in liver tissue
Although it is extremely important to examine the presence or absence of infection clinically, primers for polymerase chain reaction (PCR) can be prepared from the NANBV cDNA of the present invention and used for diagnosis of NANB hepatitis. That is, as described in (1) of Example 1, RNA was purified from 1 ml of various sera derived from patients and healthy persons. Similarly, as described in Example 1 (2), RNA was prepared from liver cells. Then, PCR and electrophoresis were performed as described in (4) of Example 1, and identification of whether the amplified cDNA by Southern hybridization was derived from NANBV was performed in accordance with a conventional method.
This was performed using a ³² P-labeled probe prepared from cDNA derived from ANBV cDNA clone BK108. Fifth
As is evident from the results shown in the table, the primers prepared from the nucleotide sequence of NANBV cDNA obtained by the present invention and the cloned NANBV cDNA fragments were used as probes to obtain NANB in serum.
V nucleic acid can be detected to diagnose serum NANBV infection.

[0104]

[0105]

Actions and effects of the invention

(1) The NANBV gene cDNA of the present invention has extremely high reliability and encompasses the entire region of the open reading frame of the NANBV gene. (2) The NANBV antigen polypeptide of the present invention is
Since it specifically reacts with BV, the diagnosis of NANB hepatitis and the case where the NANBV antigen polypeptide is used as a diagnostic agent can be easily performed with high reliability. (3) When the NANBV antigen polypeptide of the present invention is used for blood transfusion screening and when NANBV-infected blood can be easily selected with high reliability and removed from non-infected blood, Type NANB
Hepatitis can be prevented. (4) The NANBV antigen polypeptide of the present invention comprises NAN
It can be advantageously used as an active factor of a vaccine for preventing hepatitis B. (5) The NANBV antigen polypeptide of the present invention
An antibody specific to ANBV, especially a monoclonal antibody,
The antibody can be easily prepared, and the antibody specific to NANBV can be advantageously used not only as a diagnostic agent for detecting hepatitis NANB but also as an agent for removing NANBV from blood for transfusion. (6) Furthermore, it should be noted that the NANBV antigen polypeptide of the present invention is produced by gene expression of DNA encoding the antigen polypeptide present in the host cell, not by virus infection of the animal. The possibility of infection during the production phase of the antigenic polypeptide is substantially eliminated and the production costs can be reduced, and all the substances used in the production process, for example the culture medium used in the culture system, Because it is well known, it is easy to purify and it is possible to obtain a high-purity antigen polypeptide product.

[Brief description of the drawings]

FIGS. 1-1 and 1-2 show the relationship between the cDNA clones of the NANBV gene of the present invention with respect to the entire region of NANBV.

FIGS. 2-1 to 2-16 show the NANB of the present invention.
The nucleotide sequence of the entire region of the V gene cDNA and the amino acid sequence encoded by the nucleotide sequence are shown.

FIG. 3 shows a comparison of a hydrophobicity index (hydrophobic / hydrophilic pattern) between the NANBV gene cDNA of the present invention and both proteins encoded by the Japanese encephalitis virus (JEV) gene. The horizontal axis is amino acid No. , The vertical axis is the hydrophobicity index,
Open triangles indicate sugar chain-binding sites, and asterisks indicate amino acid sequences common to RNA polymerases (Gly-Asp-As
p) Sites, C, M, E and NS mean core protein, matrix protein, envelope protein and non-structural protein, respectively.

──────────────────────────────────────────────────続 き Continued on the front page Accession number of microorganism FERM BP-2972 Accession number of microorganism FERM BP-2973 Accession number of microorganism FERM BP-2974 Accession number of microorganism FERM BP-2975 Accession number of microorganism FERM BP-2976 No. FERM BP-3243 Accession number of microorganism FERM BP-3384 Accession number of microorganism FERM BP-3385 Accession number of microorganism FERM BP-3386 Accession number of microorganism FERM BP-3388 Accession number of microorganism FERM BP-3388 (72) Chisato Mori 2-9-1, Yawata-cho, Kanonji-shi, Kagawa Prefecture Osaka University Research Institute for Microbial Diseases Kannonji Laboratory (72) Invention Person Akihisa Takamizawa 2-9-14-1 Yawata-cho, Kanonji-shi, Kagawa Prefecture Inside the Kannonji Institute, Research Institute for Microbial Diseases, Osaka University (72) Inventor Iwao Yoshida 2-9-141, Yawata-cho, Kanonji-shi, Kagawa Prefecture Osaka University Microbiological Diseases Study Group Kannonji Laboratory (56) References Special Table Hei 2-500880 (JP, A) Special Table Hei 4-504715 (JP, A) Special Table Hei 5-500155 (JP, A) Proc. Japan. Acad. , 65B (1989) p. 219-223 (58) Fields investigated (Int. Cl. ⁷ , DB name) C12N 15/09 A61K 39/29 C07K 14/18 G01N 33/569 G01N 33/576 BIOSIS (DIALOG) GenBank / EMBL MEDLINE (STN)

Claims (13)

(57) [Claims] 1. The method according to claim 1 , wherein the nucleotide numbers shown in FIG.
Total salts of No. 1 to No. 9416 non-A non-B hepatitis virus
Base number 333 to 422, 333 in the base sequence
From No. 677 to No. 333 to No. 1499, No. 3
No. 33 to No. 6371, No. 474 to No. 563,
No. 678 to No. 905, No. 906 to No. 953
No., No. 906 to No. 1499, No. 1020 to No.
No. 1046, No. 1020 to No. 1121, No. 119
4th to 1232th, 1209 to 1322
No., from No. 1500 to No. 2519, From No. 2520
No. 3350, No. 3351 to No. 5177, No. 44
No. 85 to No. 4574, No. 5178 to No. 5918
No., from No. 5544 to No. 5633, From No. 5919
No. 6371, No. 6372 to No. 9362, and
Selected from the group consisting of the 1st to 9416th nucleotide sequences
A first base sequence and a second base sequence complementary to the first base sequence
At least one selected from the group consisting of two base sequences
Includes base sequence or reduces first base sequence
Both of which are obtained by replacing one base by the degeneracy of the gene codon.
Isolated at least one base sequence
Deoxyribonucleic acid. 2. A non-A non-B type shown in FIGS. 2-1 to 2-16.
Nucleotide Nos. 333 to 333 of the entire nucleotide sequence of hepatitis virus
No. 422, No. 333 to No. 677, No. 333
No. 1499, No. 333 to No. 6371, No. 474
From No. 563 to No. 678 to No. 905, No. 90
No. 6 to No. 953, No. 906 to No. 1499, No.
No. 1020 to No. 1046, No. 1020 to No. 11
21st, 1194th to 1232th, 1209th
No. 1322, No. 1500 to No. 2519, No.
No. 2520 to No. 3350, No. 3351 to No. 51
77th, 4485th to 4574th, 5178th
No. 5918, No. 5544 to No. 5633, No.
No. 5919 to No. 6371, No. 6372 to No. 93
No. 62 and the nucleotide sequence from No. 333 to No. 9362
A sequence encoded by a base sequence selected from the group consisting of
An isolated antigenic polypeptide containing a amino acid sequence. 3. A method comprising the steps of :
Selectable replicable expression vector and deoxyribonucleic acid
2-1. The deoxyribonucleic acid is shown in FIG.
Non-A non-B type of base numbers 1 to 9416 shown in No. 6
Base number 333 of the entire base sequence of hepatitis virus
No. 422, No. 333 to No. 677, No. 333
No. 1499 to No. 474 to No. 563, No. 6
No. 78 to No. 905, No. 906 to No. 953, No.
No. 906 to No. 1499, No. 1020 to No. 104
No. 6, No. 1020 to No. 1121, No. 1194
No. 1232, No. 1209 to No. 1322, No. 1
500th to 2519th, 2520th to 335th
No. 0, No. 3351 to No. 5177, No. 4485
No. 4574, No. 5178 to No. 5918, No. 5
No. 544 to No. 5633, No. 5919 to No. 637
Nucleotide sequence Nos. 1 and 6372 to 9362
A duplicable set characterized by being selected from the group consisting of
Transformation. 4. Hybridization or polymerase
Non-A non-B hepatitis detected by Zechein reaction
As shown in Figs. 2-1 to 2-16,
Non-A, non-B hepatitis C base numbers 1 to 9416
Of the entire base sequence of irs, base number 333 to base number
No. 422, No. 333 to No. 677, No. 333
No. 1499, No. 333 to No. 6371, No. 474
From No. 563 to No. 678 to No. 905, No. 90
No. 6 to No. 953, No. 906 to No. 1499, No.
No. 1020 to No. 1046, No. 1020 to No. 11
21st, 1194th to 1232th, 1209th
No. 1322, No. 1500 to No. 2519, No.
No. 2520 to No. 3350, No. 3351 to No. 51
77th, 4485th to 4574th, 5178th
No. 5918, No. 5544 to No. 5633, No.
No. 5919 to No. 6371, No. 6372 to No. 93
From the nucleotide sequence of No. 62 and Nos. 1 to 9416
First base sequence and first base sequence selected from the group consisting of
Selected from the group consisting of the second base sequence complementary to the sequence
Isolated deoxy containing at least one base sequence
Hybridization or polymerase to ribonucleic acid
Characterized by containing sufficient amount in Zechein Reaction
Diagnostic agent. 5. Detection of non-A non-B hepatitis by antigen-antibody reaction
As a diagnostic agent for informing, Fig. 2-1 to Fig. 2-16
Nucleotide number of the entire nucleotide sequence of non-A non-B
No. 333 to No. 422, No. 333 to No. 677,
333th to 1499th, 333th to 637th
No. 1, 474 to 563, 678 to 9
No. 05, No. 906 to No. 953, No. 906 to No.
No. 1499, No. 1020 to No. 1046, No. 102
0th to 1121th, 1194th to 1232
No., from No. 1209 to No. 1322, From No. 1500
No. 2519, No. 2520 to No. 3359, No. 33
No. 51 to No. 5177, No. 4485 to No. 4574
No., from No. 5178 to No. 5918, From No. 5544
No. 5633, No. 5919 to No. 6371, No. 63
No. 72 to No. 9362, and No. 333 to No. 93
According to a nucleotide sequence selected from the group consisting of the nucleotide sequence of No. 62
An isolated antigenic antigen having an encoded amino acid sequence
Contains sufficient amount of the repeptide to produce an antigen-antibody reaction.
A diagnostic agent characterized by the fact that: 6. The base number shown in FIG. 2-1 to FIG. 2-16.
Total salts of No. 1 to No. 9416 non-A non-B hepatitis virus
Of the base sequence, base numbers 333 to 422,
333th to 677th, 333th to 1499th
No., No. 333 to No. 6371, No. 474 to No. 5
No. 63, No. 678 to No. 905, No. 906 to No.
No. 953, No. 906 to No. 1499, No. 1020
No. 1046 to No. 1020 to No. 1121, No.
1194 to 1232, 1209 to 13
# 22, # 1500 to # 2519, # 2520
No. 3350, No. 3351 to No. 5177, No.
4485 to 4574, 5178 to 59
No. 18, No. 5544 to No. 5633, No. 5919
No. 6371, No. 6372 to No. 9362,
And the group consisting of the nucleotide sequences from No. 1 to No. 9416
Using at least one selected base sequence as a template
RNA synthesized by the method. 7. The non-A non-B hepatitis virus gene cDNA
Escherichia coli BK108 which is possessed
issue). 8. The non-A non-B hepatitis virus gene cDNA
Escherichia coli BK129 possessed by
issue). 9. The non-A non-B hepatitis virus gene cDNA
E. coli BK138
issue). 10. The non-A, non-B hepatitis virus gene cDNA.
Escherichia coli BK153 carrying microbes
issue). 11. The non-A non-B hepatitis virus gene cDNA
Escherichia coli BK157 carrying microbes
issue). 12. A non-A non-B hepatitis virus gene cDNA.
Escherichia coli BK166 carrying microbes
issue). 13. A non-A, non-B hepatitis virus gene cDNA.
Escherichia coli BK172 carrying microbes
issue).