JPH07258290A - Non-a non-b type hepatitis virus particle - Google Patents

Abstract

PURPOSE:To obtain the subject new type hepatitis virus particles useful as a vaccine for non-A non-B type hepatitis and a diagnostic having high detection accuracy, containing an antigen such as a core antigen of non-A non-B type hepatitis virus, a matrix antigen, an envelope antigen, etc. CONSTITUTION:The new non-A non-B type hepatitis virus particles contains at least one antigen of a core antigen of non-A non-B type hepatitis virus, a matrix antigen, an envelope antigen, etc., and is useful as a vaccine for non-A non-B type hepatitis showing high immunogenicity, a diagnostic having high antibody positive ratio and high detection accuracy and a material for researching liver diseases and hepatoma. These virus particles are obtained by selecting a clone containing a cDNA encoding the objective antigen from a cDNA group prepared from a non-A non-B type hepatitis virus genome RNA fragment, cutting out the cDNA, linking the cDNA to a manifestation vector composed of a viral gene, culturing the prepared recombinant virus and manifesting.

Description

Detailed Description of the Invention

[0001]

FIELD OF THE INVENTION The present invention relates to non-A non-B hepatitis virus particles and a method for producing the same. For more details,
Non-A non-B obtained by expressing the entire genome region of non-A non-B hepatitis virus, ORF entire region, or each nucleotide sequence region consisting of ORF region excised from NS4 and / or NS5
TECHNICAL FIELD The present invention relates to hepatitis C virus particles and an effective production method thereof. The non-A non-B hepatitis virus particle of the present invention comprises a non-A non-B hepatitis vaccine containing the same as an active ingredient, a diagnostic agent for non-A non-B hepatitis, and screening of blood for transfusion to prevent posttransfusion hepatitis. Agents are also useful to provide polyclonal or monoclonal antibodies made using non-A non-B hepatitis virus particles. That is, the non-A non-B hepatitis virus particles of the present invention are used for the production of vaccines and immunoglobulins, the production of immunological diagnostic agents, and the production of NANBV removing agents in blood for transfusion by affinity column chromatography using NANBV antibodies. It can be used in fields such as.

[0002]

[Prior art and its problems]

[Definition of non-A non-B hepatitis virus]: As is well known, viral hepatitis is a liver disease caused by infection with hepatitis virus, and the pathogenic viruses are currently A, B, and D (delta). ) Each hepatitis virus has been isolated and identified. In addition, hepatitis D virus (hepatitis delta virus)
It is a defective virus that cannot proliferate by itself, and its growth always requires hepatitis B virus as a helper virus, so that it exists only in hepatitis B patients. However,
In 1974, it was first reported that there were a large number of cases of viral hepatitis that were not due to both infections of hepatitis A and B viruses. Since then, research on this has been energetically advanced all over the world, and as a general term for such hepatitis cases, non-A
The term non-B (non-A, non-B) hepatitis has come to be used. It is known that there are multiple non-A non-B hepatitis viruses, and according to reports to date, non-A non-B hepatitis viruses have been reported.
Non-hepatitis B virus has the following 2
It is divided into two types: epidemic hepatitis or enterally transmitted through water and food.
-Transmitted non-A, non-B)
Type; and blood transmission that occurs mainly through blood and occurs frequently after blood transfusion [blood-transmit]
ted non-A, non-B] type. Of these non-A non-B hepatitis viruses, only the orally infectious non-A non-B hepatitis viruses that have become vulnerable to Africa, India, Southeast Asia, etc. have already been identified virologically. A non-B hepatitis virus has not yet been identified. Therefore, the blood-transmitted non-A non-B hepatitis is simply abbreviated as “NANB hepatitis”, and the blood-transmitted non-A non-B hepatitis virus is simply abbreviated as “NANBV”.

[Current status and problems of NANB hepatitis research]: N
Currently, research on epidemiology, clinic, diagnosis, treatment, prevention, etc. of hepatitis ANB is currently in widespread use in various parts of the world from the viewpoint of viral hepatitis. It is being actively promoted by making full use of knowledge and technology such as physics, immunology, and molecular biology (Japanese Medical Bulletin, No. 3320, pp. 3-10, 1987;
History of Medicine, 151 (13), 735-923, 19
89; Hepatobiliary-pancreas, 21 (1), 5-113, 1990; Experimental medicine, 8 (3), 201-233, 1990). For example, various reports on hepatitis NANB have been reported as follows:

(B) Epidemiology: The number of NANB hepatitis infections in Japan is estimated by the Ministry of Health and Welfare to be 60% of chronic hepatitis patients (about 720,000), 40% of cirrhosis patients (about 100,000), and It accounts for 40% (about 7,000) of patients with liver cancer, and the number of deaths due to this reaches 16,000 each year. In the United States, 150,000 to 300,000 cases of post-transfusion hepatitis occur annually, 90% of which are NANB hepatitis.
6% are considered to be NANBV carriers. Since it is estimated that the incidence rate and carrier rate are similar or higher in other countries, the establishment of diagnosis, treatment and preventive measures for hepatitis NANB brings the long-awaited gospel to the world.

(B) Virology: Previously reported NA
NBV has an enveloped spherical shape with a diameter of about 50 nm and is considered to be a virus of a type similar to Togavirus and Flavivirus in virus taxonomy. Based on the presence or absence of cytoplasmic tubular structure formation in chimpanzee hepatocytes intravenously inoculated with hepatitis NANB patient serum, pathological findings of nuclear particle appearance, epidemiology, chloroform sensitivity, immunological diagnosis, etc., 1
The existence of NANBV of one species or two or more species is estimated (Science, 205 , 197-200, 197).
9; Journal of Infectious Di
Sease, 148 , 254-265, 1983; Microorganisms, 5 (5), 463-475, 1989). In particular, A
The amount of NANBV in the blood of patients is extremely low compared with both hepatitis B and hepatitis B [Chimpanzee infecti
House doses (CID / ml) is 10 ^{8 for} B type
-10 ⁹ and 10 ⁴ -10 ⁵ for NANB] (Bra
dley DW (1985): Research pe
rspectives in posttransfu
sion non-A, non-B hepatiti
s. In DoddRY, Barker LF (ed
s): "Infection, Immunity an
d Blood Transfusion. "New
York: Alan R. Liss, Inc. , Pp
81-97), and non-human infection experimental systems.
In the present situation that only chimpanzees in which a typical intracytoplasmic tubular structure appears due to ANBV infection are known as susceptible animals, this basic research requires a large number of rare and expensive chimpanzees, and the supply restrictions on their availability. Economic constraints make it difficult and delay the critical infection experiments, identification, and marker discovery of NANBV. Therefore, new research strategies are being attempted to overcome this situation. For example, from the plasma of chimpanzees with NANB hepatitis, N called hepatitis C virus (HCV)
Clone ANBV gene cDNA (Science
e, 244 , 359-362, 1989), and the antigen called C-100 obtained by expressing the gene is NAN.
Confirmation of antigen-antibody reaction with blood antibody in hepatitis B patients (Science, 244 , 362-364, 198).
9); N without using chimpanzee as in the above example
NANBV gene cDNA directly from plasma of ANB hepatitis patients
It was confirmed that the antigen obtained by cloning A and expressing such a gene causes an antigen-antibody reaction with the antibody of the serum of NANB hepatitis patients (Gastroenterology
Japana, 24 , 540-544, and 54.
5-548,1989) and the like are known. Also, NAN
Regarding cloning of BV gene cDNA and determination of its base sequence and amino acid sequence, clones of the following institutions are known: Mitsubishi Kasei Co., Ltd. (European Patent Publication No. 29).
3274), Chiron (US) (European Patent Publication Nos. 318216, 388232 and 39874).
8), the Foundation for Research on Microbial Diseases of Osaka University (European Patent Publication No. 363025, and Journal of Vi).
Rology, 65 , 1105-1113, 199
1), Sanwa Chemical Laboratory Co., Ltd. (JP-A-1-18699)
0), National Cancer Center Research Institute of Japan [Proceed
ings of the National Acade
my of Sciences (USA), 87 , 95
24-9528, 1990], Jichi Medical School (Japan
nese Journal of Experimen
tal Medicine, 60 , 167-177, 1
990), Japan National Institute of Preventive Health [Nucleic
Acid Research, 17 (24), 1036.
7-10372, 1989; ibid., 18 (15), 46.
26, 1990; Gene, 91,287-291,1.
990; and Journal of General
Virology, 71 , 3027-3033, 199.
0) etc. Furthermore, regarding the structure of the NANBV gene, the following has now been reported: The NANBV genome has a total length of about 10 kb; the genome is 5'-terminal non-co.
ding area, open reading frame
(ORF) region and non-coding at the 3'end
The ORF region is composed of a viral core antigen protein (C antigen), a matrix antigen protein (M antigen), an envelope antigen protein (E antigen), and 6 kinds of non-structures in the order of 5 ′ to 3 ′ end. Genes encoding proteins (NS proteins) are lined up; NS protein genes are NS1, NS2, NS3, NS in order from the 5'end to the 3'end.
4a, NS4b, and NS5 genes. still,
From a functional aspect, C protein protects genes, E protein infects infection, M protein maintains structure of E protein, NS1 as complement fixing antigen, NS3 as protease, NS5 as polymerase, and non- It is considered that the coding region is involved in or acts on the structural maintenance and replication of the gene, respectively. Both NS2 and NS4 functions are currently unknown.

(C) Clinical: In general, hepatitis is epidemic hepatitis or sporadic hepatitis depending on the population and frequency of occurrence, and acute hepatitis, fulminant hepatitis, subacute hepatitis, persistent hepatitis based on severity and disease stage. , Chronic hepatitis, etc. NANB hepatitis has an incubation period of 2 to 26 weeks, and its initial symptoms are mild with fever and malaise, etc., and 70% milder than hepatitis B.
Since it has no jaundice, it is likely to be overlooked. But N
The characteristic of ANB hepatitis and its fear are that it tends to become chronic, and further, it shifts to cirrhosis. For example, NA in which increased aminotransferase activity in serum was observed
Among the cases of hepatitis NB, chronicity occurs at a rate of 40 to 50%, and 10 to 20% of the cases that become chronic are cirrhosis. In addition, 0.5 to 1% of blood recipients per year develop into cirrhosis without subjective symptoms. Unfortunately, further progress is thought to lead to hepatocellular carcinoma or liver cancer. Therefore, the eradication of NANB hepatitis is extremely important worldwide from the viewpoint of not only blood transfusion but also public health biohazard prevention related to bleeding.

(D) Diagnosis: As mentioned above, NANBV has not been identified yet, and no reliable viral marker for diagnosis is known. Therefore, finally, both hepatitis A and B,
Exclusion diagnosis based on the test of antibody titers of known respiratory viruses such as cytomegalo, EB, chickenpox, and herpes simplex, and histopathological diagnosis by liver biopsy are inevitable (S.
"Disease of the" by Shenlock
live and biliary system ”,
Eighth Edition, Pages 326-333, Blackwell
Scientific Publications 1
Published 989). In parallel with these, for example, serum enzyme, GPT [glutamate pyruvate transaminase; alias: ALT (alanine aminotransferase)], GOT [(glutamate oxaloacetate transaminase; alias: AST (aspartate aminotransferase)], guanine deaminase Measurement of increase in activities such as (alias: guanase) (hepatobiliary and pancreas, vol. 14,
Pp. 519-522, 1987); Diagnostic criteria based on the continuation of abnormally high GPT or GOT activity in serum as described above (Journal of Japanese Society of Transfusion, 31 (4), 316-).
320, 1985; Nippon Rinjo, 46 , 2638, 198.
8) etc. are adopted. Regarding immunological diagnosis, in the present situation where isolation and identification of NANBV is difficult as described above, it can be obtained by expressing a clone of the isolated NANBV cDNA by making full use of experience, technology and knowledge of genetic engineering and immunology. Antigen-antibody reaction between the antigens and NANB hepatitis patient serum has been adopted. For example, as an antigen, NANBVcDN derived from hepatitis NANB patient plasma
A expression product (European Patent Publication No. 363025), N
HCV cD derived from chimpanzee plasma with ANB hepatitis
NA expression product (European Patent Publication No. 318216 or Japanese Patent Publication No. 2-500880), NANBV cDNA expression product derived from NANBV-infected chimpanzee liver (European Patent Publication No. 293274, or JP-A-64-2576).
And JP-A-1-124387) and the like, and the reaction system includes RIA (radioimmunoassay), E
IA (enzyme immunoassay) and the like are commonly used. However, the antigenicity of such expression products has little commonality with each other, and, for example, in the case of the above-mentioned HCV C-100 antigen, it exhibits antigenicity, although it serves as a tentative index and standard of chronic hepatitis due to HCV infection. The reaction area is narrow, and the antibody detection rate is about 70%, which is insufficient (microorganisms, 5 ,
463-475, 1989; hepatobiliary pancreas, 20 , 47-5.
1, 1990; History of Medicine, 151 , 871, 198.
9) It is considered to be insufficient for definitive diagnosis of NANB hepatitis or NANBV infection in a broad sense, progress and treatment of chronic hepatitis, and determination of acute hepatitis. Therefore, further development of more detailed virological and immunological investigations is expected in order to ensure diagnosis and determination of course and treatment.

(E) Treatment and prevention: Recently, the usefulness of α and β interferons in the treatment of chronic NANB hepatitis has been reported (hepato-biliary-pancreatic, 20 , 59-64, 19).
90; History of Medicine, 151 , 871-876, 198
9). However, the dose and administration period have not yet been determined. Further, regarding a preventive agent for NANB hepatitis, for example, a vaccine, the above-mentioned NANBV cDNA expression product (European Patent Publication No. 363025) or H is used.
CV cDNA expression product (European Patent Publication No. 3182)
No. 16) and the like are known as vaccine antigens. However, under the present circumstances where NANBV itself has not been determined, a vaccine antigen is specified from the above expression products having various antigenic determinants (epitope), and the efficacy and safety of such a specific antigen are clinically sufficiently determined. The NANBV vaccine has not reached the commercial range because it requires more detailed study and time.

(V) Production of NANBV particles and their significance:
As described above (b), although various NANBV cDNA clones are known, N using such clones is still unknown.
No successful example of ANBV viral particle production is known.
This is essential for the expression of NANBV particles.
This means that it is extremely difficult to construct a cDNA of about 10 kb that covers the entire region of the BV genome. That is, NAN
In the conventionally known technique for selecting the material for extracting the BV genomic RNA and for extracting and purifying the RNA, the number is 100 at most.
Only RNA fragments as short as bases and their cDNA clones can be isolated. Therefore, in order to construct a cDNA for the entire NANBV genome region using such a short-chain cDNA fragment, the ORFs of several tens or more kinds of cDNA fragments must be matched with each other and must be linked accurately. . Needless to say, the linkage operation of cDNA fragments under such conditions is not only a very complicated and difficult technique, but especially, the probability of fatal linkage error of cDNA fragments is It increases in proportion. Therefore, in order to achieve accurate cDNA construction of the entire NANBV genome region, it is necessary to reduce the number of linkage operations using long-chain cDNA fragments, which is premised on the fact that the number of bases is about 2 k.
It requires a high degree of knowledge and experience in extracting and preparing a long-chain NANBV genomic RNA fragment of b to about 5 kb and cloning of its cDNA, and an unskilled skill. On the other hand, as described above (d), the antigen of the currently marketed NANB hepatitis diagnostic agent is an expression product of a partial fragment of NANBV genomic cDNA and has a narrow antigen spectrum. The antibody detection rate is about 70%, which is not sufficient. Therefore, not only chronic but also acute NA
It is expected that a diagnostic agent that exhibits excellent antigen-antibody reaction with sera from patients with hepatitis NB and has a high detection rate of antibodies will be put to practical use. In order to meet this expectation, it is desired to use NANBV particles having a broad antigen spectrum as the antigen of the diagnostic agent to enhance the antibody detection rate. Furthermore, it is considered that the production of NANBV particles contributes to the solution of the above problem (e) and ensures the practical application of the NANB hepatitis vaccine. As is clear from the above, NANB
Construction of cDNA of the entire V genome region and expression of N
Achieving mass production of ANBV particles is highly anticipated at the world level.

[0010]

The inventors of the present invention have conducted extensive studies to overcome the above-mentioned problems of the prior art, and as a result, 1
NANBV gene cDNA having a base number of 000 or more
The ORF region of the NANBV genomic cDNA from the C protein gene to the NS3 gene is linked to each other by linking 10 or less clones with their ORFs matched with each other, a region covering the longest NANBV total ORF longer than the region, and the entire ORF. Both non-cods at the 5'and 3'ends in the region
The NANBV genome whole region linked with the ing region was constructed accurately, and, surprisingly, the mass production and the acquisition of NANBV particles were achieved by inserting and expressing each such region in an expression vector. The NANBV particle referred to in the present invention means an expression product of the above region of the NANBV genome, and contains at least one antigen selected from the group consisting of non-A non-B hepatitis virus core antigen, matrix antigen and envelope antigen. . Examples of non-A non-B hepatitis virus (NANBV) particles include the following structures: NANB mainly composed of C (core) antigen, M (matrix) antigen and E (envelope) antigen.
V antigen assembly, NANBV complete particle having nucleic acid inside virus particle; mainly C antigen, M antigen and E
NANBV antigen assembly consisting of antigens, NANBV incomplete particles having no nucleic acid inside virus particles;
NANBV antigen assembly mainly composed of C antigen, NANBV core having nucleic acid inside its core; NANBV antigen assembly mainly composed of C antigen, NANBV incomplete core not having nucleic acid inside its core; and mainly NANBV surface antigen assembly consisting of E antigen. Such an achievement is directly (a
U.S.A.) for the purpose of extracting RNA, NANBV and its gene can be stored in a body fluid or blood without undergoing the amplification process of NANBV using a chimpanzee having an unknown transfer factor as a host and under the preservation of the gene isolation material. This is because the careful operation was carried out so as not to be cleaved / degraded by the enzyme therein and to obtain complete NANBV genomic RNA or a long-chain RNA fragment of about 2 kb to about 5 kb. In other words, the NANBV gene is cloned directly from fresh whole blood of a NANB hepatitis patient and excised liver strips of a liver cancer patient associated with NANB hepatitis, that is, a highly reliable human-derived fresh material containing the NANBV gene. Moreover, the expression was successful.
Furthermore, since the blood antigen amount of NANBV is remarkably low, which is about 10,000 times lower than that of both hepatitis A virus and hepatitis B virus, the problem that isolation and identification of the NANBV antigen is difficult was successfully solved. . That is, as described in detail later, after extracting and purifying RNA from the above-mentioned fresh material of human origin that has been strictly selected and collected, a cDNA library of such RNA is prepared using reverse transcriptase, and then the cDNA is prepared. Each cDNA from the library was cloned into a phage vector lambdagt
11 and each cDNA is expressed on a phage plaque at a high concentration, and the NANBV antigen is repeatedly detected by EIA using the expression product and acute NANB hepatitis patient convalescent serum and chronic NANB hepatitis patient serum. Have successfully screened a long-chain NANBV gene cDNA having about 1000 to about 5000 bases. Also, the NANBV genomic cDNA whole region and the NANBV genomic cDNA ORF whole region constructed by matching and linking the ORF whole regions of the cDNAs carefully selected from the cloned cDNA group are inserted into an expression vector and expressed. Because of this, N is
It was also found that the ANBV particle or NANBV antigen assembly can be obtained in large quantities and at low cost. In addition, the expression product of the present invention has a broader antigen spectrum than the expression products of conventional partial fragments of NANBV genomic cDNA, and is excellent in not only chronic but also acute NANBV hepatitis patient sera and specific antigen-antibody reaction. Therefore, it was found that the antibody detection rate is 95% or more, which solves all of the above problems (f). That is, it brings about a great progress in enhancing the immunogenicity of the vaccine, improving the detection rate of the antibody of the diagnostic agent, and producing the antibody. The present invention has been completed based on these findings. Therefore, it is an object of the present invention to provide isolated NANB hepatitis virus particles useful as an active ingredient of a diagnostic agent and vaccine for NANB hepatitis. The present invention also aims to provide a method for producing isolated NANBV particles. or,
The present invention aims to provide a diagnostic agent for NANB hepatitis. Moreover, this invention aims at provision of the vaccine of NANB hepatitis.

That is, according to the present invention, an isolated non-A non-B hepatitis virus containing at least one antigen selected from the group consisting of core antigens of non-A non-B hepatitis virus, matrix antigens and envelope antigens. Viral particles are provided.

More specifically, the core antigens, matrix antigens and envelope antigens of the NANBV particles of the present invention are non-A non-B type having the nucleotide sequences Nos. 1 to 9416 shown in FIGS. 2-1 to 2-16. 333 to 677, 678 to 905 of the entire nucleotide sequence of hepatitis virus
Nos. And 906 to 1499th base sequences, respectively.

According to another aspect of the present invention, FIG.
To non-A non-B hepatitis virus particles having ribonucleic acid corresponding to at least a part of the nucleotide sequences shown in FIGS.

In the present invention, unless otherwise specified, the left and right ends of the deoxyribonucleic acid sequence are the 5'end and the 3'end, respectively. Unless otherwise specified, the left and right ends of the amino acid sequence of a peptide are the N-terminus and C-terminus, respectively. Isolated NANBV of the present invention
Particles can be prepared and identified according to steps (I) to (VII) below. (I) Selection and collection of RNA extraction material of NANBV gene: NANBV RNA extraction material is NANB
V carriers or blood, lymph, ascites, hepatocytes, and NA of humans or chimpanzees affected by them
Hepatocellular carcinoma associated with hepatitis NB or hepatocytes of liver cancer patients can be used. Especially in the chimpanzee-derived material, N
The amount of ANBV is relatively small compared to that of humans, and NA
The use of human-derived material is desirable because it contains unknown factors that are considered difficult to separate NBV. blood,
Of the lymph, ascites, and hepatocytes, blood is most easily obtained in large quantities, for example, blood that cannot be used as blood for transfusion is available in large quantities from blood banks. Such blood can be advantageously used as a NANBV RNA extraction material. 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 the surface antigen of the hepatitis B virus (WHO expert committ).
ee on viral hepatitis: Adv
ances in viral hepatitis,
WHO Technical Report Seri
es, 602 , 28-33, 1977) and the gene DNA of hepatitis B virus (Brechot, C; Ha.
dchouel, M; Scotto, J; Degos,
F; Charnay, P; Trepo, C; Tioll
ais, P: Detection of hepati
tis B virus DNA in liver
and serum: a direct appraisal
sal of the chronic carrier
r state. Lancet, 2 , 765-768,
1981) and determine whether the plasma is a serum enzyme that has been adopted as a standard for the diagnosis of hepatitis NANB, such as GPT (Wrobleski,
F. & LaDue, J .; S. : Serum glut
amic-pyruvic transaminese
in cardiac and hepatic di
sease, Proc. Soc. Exp. Biol. M
ed. , 91 , 569, 1956. ), GOT, guanase, etc. The above-mentioned step of separating blood into plasma and red blood cells and inspecting the plasma is carried out for various bloods, and both the surface antigen of hepatitis B virus and gene cDNA are negative, and the activity of the enzyme is abnormally high. , For example, pool plasma with a GPT activity of 35 IU / l or more.

The number of NANB hepatitis virus particles in the blood is
It is very small compared to the number of hepatitis B virus particles described above. From the results of infection experiments (Bradley, DW: Res
search perspectives in pos
t-transfusionnon-A, non-B
hepatitis: “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 / 10,000 of the number of hepatitis B virus particles, the RN
It is desirable to use a large amount of blood, for example about 3 to 10 l, for the extraction of A. This fresh whole blood was used as a material for extracting NANBV RNA from virus particles,
In order to avoid denaturation of NBV and its gene and gene cleavage / degradation by enzymes, it is stored at 1 to 5 ° C., and NANBV RNA is prepared by the following step (II) within 48 to 72 hours after collection of fresh whole blood. It is desirable to complete. . When hepatocytes are used as a material, NAN
B Approximately 1 to 3 pieces of resected liver tissue pieces of a non-cancerous part and a cancerous part obtained from a patient with hepatocellular carcinoma or liver cancer associated with chronic hepatitis
g can be used advantageously. Hepatocytes used as a material are stored frozen, for example, at -70 ° C.

(II) Preparation of RNA of NANBV: From the material obtained in (I) above, RNA can be extracted and purified by known means. For example, when fresh whole blood is used as a material, about 2 to 10 l of blood is subjected to low-speed centrifugation, a plasma fraction of the supernatant is collected, and a virus fraction is purified from this plasma, and then RNA extraction described below is performed. Subject to the purification process.

On the other hand, when liver strips are used as the RNA extraction material for NANBV, a known diluent is added to the liver tissue after adding a diluent containing the following RNA degrading enzyme inhibitor in a volume of about 5 to 30 times its volume. A liver cell homogenate is obtained by crushing or grinding the liver tissue by a means such as a homogenizer. In addition, as a diluent, 10 to 150 m
Known buffer solutions of M can be used. Then, this is subjected to low speed centrifugation to collect the supernatant, and the supernatant is
Use as a stock solution for extraction and purification. For extraction and purification of RNA, a known method, for example, extraction using a mixed solution of an RNA degrading enzyme inhibitor such as heparin, diethylpyrocarbonate, guanidine thiocyanate and a surfactant, a chelating agent or a reducing agent that enhances protein denaturation Method; sucrose, cesium chloride, cesium trichloroacetic acid, ficoll [Pha
[rmicia Fine Chemicals AB (Sweden)] etc. as a solute of the gradient] Fractionation method by density gradient centrifugation; Separation method by affinity column using poly A chain at 3'end specifically possessed by mRNA; Synthesis on polysome Method for obtaining mRNA-bound polysomes by immunoprecipitation using specific antibody against the purified protein; phenol extraction method based on the principle of two-phase partitioning; precipitation method using polyethylene glycol, dextran sulfate, alcohol, etc. individually or in combination Can be used. The above-mentioned NANBV RNA extraction and purification steps were 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 NANBV RNA obtained above, cDNA can be prepared by a conventional method.
That is, oligodeoxythymidine and random hexanucleotide primers and a reverse transcriptase were used as primers, cDNA of the NANBV RNA was used as a template to synthesize a cDNA complementary thereto, and the cDNA and the NANBV RNA bound to each other complementarily. To obtain a double strand consisting of
Next, ribonuclease H is allowed to act on this to give the NA
After NBV RNA is decomposed and removed from the cDNA, double-stranded cDNA is synthesized by using the single-stranded cDNA as a template and a DNA synthase. Double-stranded cDNA synthesis can be performed using commercially available cDNA synthesis kits such as cDNA Synth.
sisSystem Plus (registered trademark) [Ame
Made by rsham (UK)], cDNA System
Kit (registered trademark) [Pharmacia LKB (Sweden)], cDNA Synthesis
Kit (registered trademark) [Boehringer Man
nheim GmbH (Germany)] and the like. When the amount of synthesized cDNA is small, the conventional PCR (Polymerase Chain)
Reaction) method (Errich, HA:
"PCR Technology, Stockton
Press, 1989), Am for PCR kit.
pliTaq [Perkin Elmer Cetus
(Manufactured by USA)]).

(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 cleaving the cDNA prepared in 1. into fragments having various lengths and ligating each of the various cDNA fragments thus obtained with a recombination cloning vector. As the recombinable cloning vector, for example, known or commercially available vectors such as phage gene, cosmid, plasmid and animal virus gene can be used. When using a phage gene or cosmid as a recombinable vector,
In order to achieve a high degree of stabilization of the respective vectors into which each cDNA fragment has been individually inserted and the appearance rate of transformants, in vitro packaging is performed by a conventional method, and each cDNA inserts the inserted vector in the form of recombinant phage particles. CDNA library as a phage particle cDNA library.
To be used for cloning. On the other hand, when a plasmid is used as a vector that can be recombined,
The NA fragment was inserted into a plasmid vector, and these cDNAs
The NA insertion vector was transformed into E. coli, Bacillus subtilis, and
The transformant obtained by transferring into a host cell such as yeast is cDNA
It serves for cloning of cDNA as a library.
When using an animal virus gene as a recombinable vector, each of the above-mentioned cDNA fragments is inserted into a virus gene vector, and each of these recombinant viruses is transfected into susceptible animal cells according to a standard method. , Grow in cells. For recombinant viruses,
The obtained recombinant virus itself is used as a cDNA library.

The cDNA library can be prepared by using commercially available kits, for example, cDNA cloning systems Lambda gt10 and Lambda gt11 [Amersham (UK), BRL (US), Stratagene (US)], in vitro packaging system. [Amersham (UK), BRL (US), S
manufactured by Tratagene (USA)] and the like.

(V) NAN from cDNA library
Cloning of cDNA containing BV gene: In this step, a cDNA clone containing NANBV gene is obtained. If the cDNA library contains transformants, these transformants are cultured in standard agar medium to form colonies. On the other hand, in the case where the cDNA library contains recombinant phage particles or recombinant viruses, these recombinant phage particles or recombinant viruses may be used for known sensitive host cells such as Escherichia coli, Bacillus subtilis, yeast and animal cell cultures. After infection, the plaques are formed by culturing or infected cells are proliferated. Then, under a known immunoassay, NA was recovered regardless of the acute NANB hepatitis convalescent serum, chronic NANB hepatitis patient serum, intrahepatic cytoplasmic tubular structure forming type or non-forming type.
By using a chimpanzee serum infected with NBV and reacting NANBV antigen-producing colonies, plaques or infected cells with at least one of the above-mentioned sera,
Select and isolate. For the strict selection of colonies, the previous steps were repeated and from each isolated colony, plaque or infected cell, T. Mole by Maniatis et al.
color Cloning, A Laborato
ry Manual (Cold SpringLabo
laboratory [U. S. A], 309-433, 198.
A cDNA clone containing the NANBV gene is isolated according to 2). The immunoassay can be performed by, for example, an enzyme-labeled antibody technique using an antibody labeled with the enzyme peroxidase or alkaline phosphatase, and a fluorescent antibody technique using an antibody labeled with fluorescein isothiocyanate or europium. In the immunoassay according to the above-mentioned technique, it is desirable to carry out the method by an indirect method capable of achieving a highly sensitive immunoassay even when a small amount of patient serum is used. In this indirect method, it is preferable to use serum of NANB hepatitis patients or NANB hepatitis chimpanzees, which have a relatively high content of antibodies specific to NANBV, as the primary antibody, and an enzyme or fluorescent substance as the secondary antibody. Labeled commercial anti-human Ig
(Immunoglobulin) antibody can be used.

Samples for immunoassay are
For example, a blotting method of adsorbing colonies, plaques or nucleic acids and proteins of infected cells on a filter membrane,
It can be prepared according to the method using a microplate or a slide glass for speculum. When the indirect enzyme antibody method is applied to the blotting method, selection of desired colonies, plaques or infected cells from a large number of original colonies, 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 N
Since it is a part of the ANBV gene, in order to obtain a cDNA clone covering the entire region of the NANBV gene, the 3'and 5'ends of the cDNA clone were used as probes, and another cDNA near the cDNA clone was used.
CDN obtained as a clone by the method of isolating the fragment
It is necessary to extend the length of the A region. In this case,
Gene Walking (Also known as the genomic walk)
ing or chromesome walking)
Can be adopted (“DNA Cloning
Volume III ", edited by DM Glover, 37.
-39, IRL Press, 1987; Mani
atis et al. , "Molecular Cl
oning-a laboratory manu
al ″ 2 , 3.21-3.23, 1989). By repeating the cloning operation and gene walking, the entire NANBV 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. The nucleotide sequence of the cDNA clone can be determined by a conventional method, for example, the Maxam-Gilbert method or the dideoxy chain terminator method (Analytical Biochemistr).
y, 152 , 232-238, 1986) and the like.

The 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 on A). For important parts of such amino acid sequence, for example, hydrophilic sites that are considered to constitute an epitope, peptides corresponding to the respective hydrophilic sites are synthesized, and after purification by high performance liquid chromatography (HPLC), these peptides are analyzed. Enzyme immunoassay (E
IA) or radioimmunoassay (RIA).

The cDNA clones are preferably classified into several groups according to the characteristics of the NANBV antigen polypeptide encoded by these clones in order to distinguish them from each other. In this connection, the positioning 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, NANBV is known to have a tubular structure-forming ability in chimpanzee liver cytoplasm, and not have it (Science, 205 , 197-20).
0, 1979), and examining the serological reactivity of each cDNA clone with chimpanzee serum infected with NANBV that forms a tubular structure in the chimpanzee liver cytoplasm, and with chimpanzee serum that does not form a tubular structure. Allows these clones to be identified and classified.
This test of serological reactivity can be performed by the immunoassay described above.

The prefix "BK" is added to the cDNA clone of the NANBV gene of the present invention shown in FIGS. 1-1 and 1-2 in the present invention. Figure 1-1 shows the NAN
It is a figure which shows the relationship between cDNA of this invention NANBV gene with respect to the whole area | region of BV gene. Further, FIG. 1-2 is a diagram showing the relationship between cDNA clones obtained by gene walking for the entire region of the NANBV gene.

The NANBV cDNA clone is, for example,
Escherichia coli BK108 (Ministry of Fine Arts, Article 2971), BK129 (Ministry of Fine Arts, Article 297)
No. 2), BK138 (Ministry of Industrial Science and Technology No. 2973), BK1
53 (Ministry of Fine Arts, Article 2974), BK157 (Ministry of Fine Arts, Article 3243), BK166 (Ministry of Fine Arts, Article 297)
No. 5) and BK172 (Ministry of Industrial Technology Article No. 2976). These 7 BK NANBVcD
The NA clone contains at least the entire open reading frame region of the NANBV gene, or perhaps N.
It is considered to cover the entire ANBV gene region.
Furthermore, in addition to the above clones, the following 5 clones have been deposited with the Institute of Micro Engineering as a representative example of the BK series: BK1
No. 02 (Microtechnical Article No. 3384), BK106 (Microtechnical Article No. 3385), BK112 (Microtechnical Article No. 338)
No. 6), BK146 (Ministry of Industrial Science, Article No. 3387), BK
147 (Ministry of Mechanical Engineering, Article 3388).

The nucleotide sequences of the entire NANBV gene region covered by the above BK NANBV cDNA clone and the amino acid sequences encoded by these 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, the homology between the nucleotide sequence and amino acid sequence of the NANBV gene and those sequences of other viral genes, and the hydrophobicity of the amino acid sequence. Various studies and observations can be made regarding sex index (hydrophobic / hydrophilic profile), structure of NANBV gene, epitope (antigenic determinant) region, and the like.

Regarding the homology, the nucleotide and amino acid sequences of the NANBV gene and various known viral genes (Japanese Patent Laid-Open No. 62-286930; Virolog) are used.
y, Vol. 161, 497-510, 1987).
Bovine viral diarrhea virus (V
irology, volume 165, pages 497-510,
1988), swine fever virus (Virology,
171, p. 555-567, 1989), tobacco leaf vein mottle virus (Nucleic Acid R)
esarch, 165, 5417-5430, 1986) and the like.

Regarding the analysis of the hydrophobicity index, for example, SDC-Genet of gene information processing software is used.
yx [made by SDC Software Inc. (Japan)], D
A program created by ooltitle (Journal
of Molecular Biology, No. 157
Vol., Pp. 105-132, 1982) and the like.

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 indexes of both viruses are compared with each other. In addition, various antigen proteins of NANB virus particles,
That is, each gene region encoding viral core antigen protein (C antigen), matrix antigen protein (M antigen), envelope antigen protein (E antigen), and 6 types of nonstructural proteins (NS proteins) is encoded by it. Hydrophobicity indices between each peptide and known flaviviruses are compared, and the amino acid sequences of such peptides are compared with signal peptidases derived from host cells (Journal of Molecular).
r Biology, 167 , 391-409, 198.
3) and a known serine protease derived from flavivirus (Virology, 171 , 637-639, 19).
89) can be determined by comparing with the peptide binding site on which 89) acts. The NANBV genome of the present invention can be shown as follows using the base numbers disclosed in FIGS. 2-1 to 2-16: C protein is the third
33 to 677, M protein is 678 to 90
No. 5, E protein No. 906 to No. 1499, NS1 No. 1500 to No. 2519, NS2 No. 2520 to No. 3350, NS3 No. 3351 to No. 517
7th, NS4a is 5178th to 5918th, NS
4b is number 5919 to 6371, and NS5 is number 6372 to 9362. It can be pointed out that each antigen protein encoded by each region of these genes is useful as an antigen for a NANB hepatitis vaccine or diagnostic agent. In addition, since the NANBV particles of the present invention have a wide variety of epitopes, a diagnostic agent using such NANBV particles or NANBV antigen assembly as an antigen is more susceptible to NANB hepatitis than a diagnostic agent having a specific epitope region as an antigen. The specific reaction range (reaction spectrum or antigen spectrum) with a wide variety of anti-NANBV antibodies induced under various conditions is wide. It turns out to be extremely high.

(VI) Expression of the entire NANBV genomic cDNA region and ORF region, and NANBV antigen assembly, NANBV incomplete particles and infectious NANB
Large-scale production of V complete particles: This step is carried out to express the NANBV gene cDNA cloned in (V) above in large amounts for industrial use. First, the entire region of each cDNA clone of the NANBV gene or a portion thereof is cut out with a restriction enzyme to obtain the NANBV gene cDNA.
After preparing the A fragment, the cDNA fragment is linked and then inserted into an expression vector. That is, NANBV genome cDNA
To construct the entire region of A and the entire ORF, clone c
In order to save labor in linking DNA fragments and to avoid linking errors, 10 or less or 5 or less cDNAs
The use of fragments is preferred. For that purpose, a long-chain cDNA having a DNA base number of 1000 or more, preferably 1500 or more
A clones are carefully selected, and the ORFs of these cDNAs are matched with each other and linked. For example, the NA disclosed in FIGS.
Among NB cDNA clones, BK112 and BK14
By using 6, BK147, BK157 and BK166, the above entire region can be constructed. Known or commercially available expression vectors, for example, plasmid vector pSN508 of the family Enterobacteriaceae (US Pat. No. 4,
703, 005), yeast plasmid vector-pBH.
103 series (JP-A-63-22098) and pJM105
(JP-A-62-286930), vaccinia virus W
R strain (ATCC VR-119), LC16m8 strain (Japanese Patent Publication No. 55-23252), attenuated chickenpox virus Oka strain (US Pat. No. 3,985,615), attenuated Marek's disease virus (Journal of Japanese Veterinary Medicine, vol. 27, 20-24 pages, 1
984; and Gan Monograph on
Cancer Research, Volume 10, 91-1
07, 1971), pTTQ series [Amers
manufactured by ham (UK)], pSLV [Pharmacia
LKB (Sweden)] and the like can be used. Then, an expression vector in which the NANBV gene cDNA is linked and linked is transferred or transfected into a host cell sensitive to the vector by a conventional method, and a transformant or infected cell producing NANBV particles can be selected and collected.
The presence or absence of NANBV antigen production can be detected in the same manner as in the immunoassay described in (V) above. Also, NA
The NBV particles can be confirmed or measured by a conventional method such as electron microscopy, immunoelectron microscopy, density gradient centrifugation or light scattering. As a result, when a plasmid was used as an expression vector, a transformant producing NANBV particles was obtained, and when an animal viral gene, that is, a viral vector was used, for example, European Patent Publication No. 334530. It is possible to obtain a recombinant virus such as that shown in
Then, such transformants and recombinant viruses are respectively cultured by a conventional method, and homogeneous NANBV is contained in these cultures.
The particles can be produced in large quantities.

(VII) Purification of NANBV particles: Purification of NANBV particles in the culture obtained in the above (I) can be carried out by a known method such as salting out, adsorption / desorption method using silica gel or activated carbon, organic solvent. Precipitation method, fractionation method by ultracentrifugation, separation method by ion exchange and affinity column chromatography, high performance liquid chromatography,
This can be carried out by appropriately combining and adopting a fractionation method by electrophoresis and the like. When NANBV particles are purified and collected from a culture of a transformant such as Escherichia coli or yeast, contamination with allergens derived from E. coli or yeast will reduce the quality of the final product. Treatment, adsorption and desorption of impurities by activated carbon, and fractionation by density gradient centrifugation are performed sequentially (JP-A-63-29).
7) is desirable. Further, when purifying and collecting NANBV particles from a recombinant virus culture, for example, a cell culture medium, highly pure NANBV particles can be collected by repeatedly performing ultracentrifugation or density gradient centrifugation. it can. Furthermore, in order to detoxify NANBV particles in the culture to ensure safety and to fix and stabilize the immunogenicity or antigenicity of the antigen, the culture before purification or after sterilization thereof A conventional inactivating agent such as formalin is added to the culture solution at a final concentration of 0.0001 to 0.001.
After adding and mixing so as to be V / V%, 5 to 9 at 4 to 37 ° C
Inactivation of NANBV antigen by storage for 0 days is desirable.
However, the recombinant attenuated virus can be used as an active ingredient of a live vaccine without inactivating it. Such a highly purified NANBV particle suspension can be used as a NANBV particle stock solution (NANBV vaccine stock solution) for the production of vaccines and diagnostic agents.

That is, according to still another aspect of the present invention,
(A) Providing 10 or less different cDNA clone groups each having a base number of 1000 or more prepared from a non-A non-B hepatitis virus genomic RNA fragment of at least 1000 bases, wherein the cDNA clone group Each cloned cDNA group as a whole has a non-A nucleotide sequence No. 1 to 9416 shown in FIG. 2-1 to FIG. 2-16.
It covers at least the region from 333rd to 5177th of the entire non-hepatitis B virus nucleotide sequence,
(B) A cDNA fragment group having a base sequence determined in advance so that the base sequence obtained when sequentially arranged has at least a region corresponding to the base numbers 333 to 5177. (C) The cDNA fragment groups each having these pre-determined base sequences are sequentially ligated to each other to obtain the non-nucleotides of the base sequences 1 to 9416 shown in FIGS. 2-1 to 2-16. A first deoxyribonucleic acid containing at least the nucleotide sequence from the nucleotide number 333 to the nucleotide number 5177 of the whole non-B hepatitis A virus sequence is constructed, and (d) the first deoxyribonucleic acid and the first deoxyribonucleic acid At least one selected from the second deoxyribonucleic acid obtained by substituting at least one base in the nucleotide sequence by degeneracy of the genetic code One deoxyribonucleic acid is introduced into a replicable expression vector selected from a plasmid and an animal virus gene, and when the replicable expression vector is a plasmid, the plasmid and the at least one deoxyribonucleic acid introduced therein are replicable Recombinant DNA, or in the case where the expression vector is an animal virus gene, a recombinant virus having the animal virus gene and at least one of the deoxyribonucleic acid introduced thereinto is used in (e) step (d). When the replicable expression vector is a plasmid, the recombinant DNA is transfected into a prokaryotic cell or a eukaryotic cell to form a transformant, and the transformant is then isolated from a prokaryotic or eukaryotic cell culture. The transformants obtained in (f) step (e) are selected and collected. And cultured in a cell, it produces non-A, non-B hepatitis virus particle by expressing the deoxyribonucleic acid, or step (d)
By culturing the recombinant virus obtained in 1. and expressing the animal virus gene and the deoxyribonucleic acid, non-A non-B
Producing hepatitis C virus particles in the form of a propagating recombinant virus comprising an animal virus and a non-A non-B hepatitis virus particle expressed therewith, and (g) the non-A non-B hepatitis virus particle alone or There is provided a method for producing non-A non-B hepatitis virus particles, characterized in that it is isolated in the form of a propagated recombinant virus.

More specifically, the above-mentioned method in which the base sequence of the first deoxyribonucleic acid consists of base numbers 333 to 5918, and the base sequence of the first deoxyribonucleic acid contains base numbers 333 to 5918. The above-mentioned method comprising the base sequence of 6371, the above-mentioned method wherein the base sequence of the first deoxyribonucleic acid consists of the base numbers 333 to 9362, and the base sequence of the first deoxyribonucleic acid has the base number There is provided the above method comprising the nucleotide sequences from 1st to 9416th.

According to still another aspect of the present invention, a replicable expression vector selected from plasmids and animal virus genes and deoxyribonucleic acid are included, and the deoxyribonucleic acid is shown in FIGS. 2-1 to 2-16. Nucleotide sequence represented by the first to 9416th non-A non-B hepatitis virus whole base sequence of at least 333 to 5177 first base sequence and at least one base of the first base sequence There is provided a recombinant characterized by being selected from the second base sequence obtained by substituting the gene codon by degeneracy.

More specifically, the above-mentioned recombinant in which the first base sequence consists of the base numbers 333 to 5918, and the first base sequence has the base numbers 333 to 6371. The above recombinant comprising a base sequence, the above-mentioned recombinant comprising a first base sequence consisting of the base numbers 333 to 9362, and the first base sequence consisting of a base sequence 1 to The above recombinant comprising the 9416th nucleotide sequence is provided.

The recombinant is the NANBV of the present invention.
The NANBV gene cDNA of the present invention can be amplified not only by production of particles but also by replication.

The purified NANBV particles of the present invention are N
It is useful as a diagnostic agent for hepatitis ANB. Further, the NANBV particles of the present invention can be prepared into a diagnostic agent as follows. Purified NANB obtained in the above (VII)
The V particle solution is dispensed into a vial or a container such as an ampoule and then sealed. The injected particles can be lyophilized by the same means as described above before sealing, and the N
The amount of ANBV particles is generally about 1 μg to about 10 mg. Alternatively, the NANBV particles can also be adsorbed to the surface of commonly used supports such as microplates, polystyrene beads, filter papers or membranes.

The determination of the reactivity between serum and the NANBV particles can be individually carried out by the same means as described in (V) above. That is, the determination of such reactivity is determined by conventional immunoassay methods, such as radioimmun.
oassay (RIA), enzyme-linked
immunosorbent assay (ELIS
A), fluorescent antibody method (FA), passive red blood cell collection reaction (PH
A), reverse passive red blood cell collecting reaction (rPHA), etc. can be used. The amount of the NANBV particles used in the above immunoassay is generally about 0.1 to about 100 mg / ml in serum, especially RIA, ELISA, F
The amounts used for A, PHA and rPHA were 0.
1-1 mg / ml, 0.1-1 mg / ml, 1-100
mg / ml, 1-50 mg / ml and 1-50 mg /
It becomes ml.

The NANBV particles 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) contacting serum of unknown blood with isolated NANBV particles, and (c) determining whether or not the serum reacts with the NANBV particles. And (d) classify positive or negative for non-A non-B hepatitis based on its reactivity, and (e)
The blood is separated according to the identification.

The contact between the serum of unknown blood and the NANBV particles of the present invention and the determination of their reactivity can be carried out by the same means as described above for the method of diagnosing NANB hepatitis. By this method, blood for transfusion that is not infected with NANBV can be selected.

The NANBV particles of the present invention are
It can be advantageously used as an active ingredient of hepatitis B vaccine.

The NANBV vaccine stock solution prepared in the above (VII) was filtered by a conventional method using a microfilter to sterilize the solution, and then the filtrate was Low with a physiological salt solution.
The protein concentration measured by the ry method is about 1 to about 500 μg /
Aluminum hydroxide gel as an adjuvant was diluted to give a final concentration of about 0.1 to 0.1 ml.
Add to about 1 mg / ml. Furthermore, at least one stabilizer can be added to such a mixture, and a commercially available known stabilizer can also be used as the stabilizer, for example, gelatin, a hydrolyzate thereof, albumin, glucose of saccharides. , Fructose, galactose,
Sucrose and lactose as well as the amino acids glycine, alanine, lysine, arginine, glutamine and the like can be used. As the adjuvant, calcium phosphate gel, aluminum phosphate gel, aluminum sulfate, alumina, and bentonite which are precipitation adjuvants, and a muramyl peptide derivative which is an adjuvant for inducing antigen production, polynucleotide, Krestin [registered trademark, Kureha Chemical Industry Co., Ltd. (Japan ) Production and sales] and Picibanil (both are antitumor agents) can also be used.

Then, the NANB hepatitis vaccine solution containing the gel-adsorbed NANBV particles was dispensed into a small container such as an ampoule or a vial and sealed, whereby the precipitated N
A purified precipitated NANB hepatitis vaccine containing ANBV particles is obtained.

By freeze-drying the thus 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, in the tropics. Such freeze-drying is generally carried out by dispensing a liquid precipitated hepatitis NANB vaccine into a vial, a container such as an ampoule and the like according to a conventional method, and after freeze-drying, nitrogen gas is injected into the container containing the dry vaccine to seal it. Regarding the quality of vaccine preparations, "Precipitation B" specified in the Japanese Ministry of Health and Welfare Notification No. 159 "Standards for Biological Products"
Test according to "Hepatitis C Vaccine", "Dried Japanese Encephalitis Vaccine" and "Precipitated Pertussis Vaccine".

Further, the NANB hepatitis vaccine can be prepared in the form of a mixed vaccine containing at least one antigen other than the above-mentioned precipitated NANBV particles and the present NANBV particles. As an antigen other than the present NANBV antigen polypeptide, side effects and adverse reactions caused by using the NANBV particles and other antigens in combination do not increase additively or synergistically, It is possible to use any antigen conventionally used as the active ingredient of the corresponding vaccines, as long as the immunogenicity is not reduced by their mutual interference.
The number and type of antigens that can be mixed in the NANBV particles are such that their side effects and adverse reactions do not increase additively or synergistically and their antigenicity and immunogenicity are reduced as described above. Unless you are not limited. Generally, it is possible to mix 2-6 antigens with the NANBV particles, such as Japanese encephalitis virus,
HFRS (renal symptomatic hemorrhagic fever) virus, influenza virus, parainfluenza virus, hepatitis B virus, dengue virus, AIDS virus, B. pertussis,
Antigens derived from diphtheria, tetanus, meningococcus, pneumococcus and the like can be used.

Generally, the vaccine containing NANBV particles of the present invention is injected into a vial such as an ampoule and sealed, and
It can be provided in the form of a liquid or suspension. When a dried vaccine is used, it is dissolved or suspended in sterile distilled water so as to have the original volume before freeze-drying before use. In general,
This vaccine is used by subcutaneous inoculation, and the amount of vaccination for an adult is about 0.5 ml, but for a child, vaccination of half the amount of an adult is vaccinated. In addition, such a vaccine is usually administered twice at intervals of about 1 week to 1 month, and then about 1 year later, another booster injection is performed.

Further, the NANBV particles can be used for preparation of antibodies such as polyclonal antibodies and monoclonal antibodies specific to them. For example, the NAN
A polyclonal antibody specific to BV particles can be prepared by the following conventional method. First, the purified NANBV particles of the present invention are added subcutaneously, intramuscularly, intraperitoneally or intravenously to animals such as mice, guinea pigs and rabbits, usually 1 to 4 times.
Inoculate several times at weekly intervals to completely immunize such animals. Conventional, commercially available adjuvants can be used to enhance the immune effect. Then, serum was collected from the immunized animal, and then anti-NANBV was collected from the serum according to a conventional method.
The particle polyclonal antibody is isolated and purified.

On the other hand, the monoclonal antibody specific to the NANBV particles is, for example, described in "Cell Engineering" Vol.
It can be prepared by a conventional method described on page 9 (1982). For example, splenocytes obtained from mice immunized with purified NANBV particles and commercially available mouse myeloma cells are fused by a cell fusion technique to prepare hybridomas, and then the hybridomas react with the NANBV particles. A hybridoma having the ability to produce an antibody is obtained. This is cultured by a conventional method, and the anti-NANBV particle monoclonal antibody is isolated and purified from the culture supernatant.

The above-mentioned polyclonal antibody and monoclonal antibody can also be used as a diagnostic agent for NANB hepatitis. NANB using such antibodies
Diagnosis of hepatitis B can be carried out in substantially the same manner as the above-mentioned method for diagnosis of hepatitis NANB using the NANBV particles. By using a polyclonal antibody or a monoclonal antibody, it is possible to identify and quantify the NANBV particles present in liver tissue and blood. Further, the polyclonal antibody and monoclonal antibody specific to the NANBV particles of the present invention can be used as a NANBV removing agent for blood for transfusion. That is,
NANBV present in blood can be effectively removed by a polyclonal antibody or a monoclonal antibody by an antigen-antibody reaction.

[0053]

EXAMPLES Specific examples of the present invention will be described below with reference to examples and reference examples. However, the present invention is not limited to the following examples. In addition, Example 1 was divided into Part I and Part II, and Reference Examples 1 to 5 were inserted between them.

Example 1 (Part I) Step 1 [DNA complementary to NANBV genomic RNA
Preparation of Plasma-Derived RNA to Produce (cDNA)] To obtain NANBV from plasma, blood GPT value (Wrob
lewski, F & J. S. LaDue: Serum
glutamic-pyruvic transam
inase in cardiac and hepa
tic disease. Proc. Soc. Exp.
Biol. Med. , 91: 569, 1956) is 3
Sucrose 30% with 4.8 L human plasma showing 5 IU / L or more
(W / W) After layering on the solution, centrifugation (48,000 xg, 1
3 hours, 4 ° C), and precipitate 50 mM Tris · HC
1, pH 8.0-1 suspended in EDTA solution,
After centrifugation (250,000 × g, 3 hours, 4 ° C.) again, the resulting precipitate was added to 75 ml of 5.5M GTC solution (5.5
M guanidine thiocyanate, 20 mM sodium citrate, pH 7.0, 0.05% sarcosyl, 0.1
M2-mercaptoethanol) and dissolved in 16 ml of CsTFA-0.1M EDTA solution (ρ = 1.51).
Layered on top. Centrifugation (140,000 xg, 20 hours,
15 ° C) to precipitate RNA. Protein and DNA in the supernatant are removed by aspiration, and the precipitate is removed with 200 μl TE (1
0 mM Tris.HCl, pH 8.0, 1 mM ED
TA) solution, add 20 μl of 3M sodium chloride and ethanol, and leave at −70 ° C. for 90 minutes. Centrifuge (12,000 xg, 30 minutes, 4 ° C), and precipitate with TE.
, Sodium chloride and ethanol were added again in the same manner as above, the mixture was allowed to stand at -70 ° C, and the resulting precipitate was recovered,
It was dissolved in 10 μl of TE to obtain purified RNA.

Step 2 (Preparation of Liver-Derived RNA for Preparation of cDNA for NANBV Genomic RNA) To obtain NANBV genomic RNA from the liver, NANB was prepared.
The method of Okayama et al. (H. Okaya)
ma, M .; Kawaichi, M .; Brownstei
n, F. Lee, T .; Yokota, and K.K. Ar
ai: High-Efficiency Clonin
go of Full-Length cDNA; Con
structure and Screening o
f cDNA Expression Librari
es for Mammalian Cells. Me
methods in Enzymology 154 . Three
-28, 1987). That is, 1 g of the excised liver was fragmented and 100 ml of 5.5 M of step 1 was used.
It floated in the GTC solution and homogenized using a Teflon-glass homogenizer. Then, the homogenate was taken in and out using a syringe with # 18 needle to mechanically cut the DNA. This homogenate is centrifuged at low speed (1,
(500 × g, 15 minutes, 4 ° C.) supernatant was layered on CsTFA solution in the same manner as described in step 1 and after centrifugation, the resulting RNA fraction of the precipitate was 0.4 ml of 4M.
Suspend in GTC solution, add 10μl of 1M acetic acid and 300μ
l of ethanol is added and the mixture is allowed to stand at −20 ° C. for 3 hours or more to precipitate nucleic acid (RNA). Centrifuge (12,000 xg,
10 minutes, 4 ° C.), dissolve the precipitate in 1 ml of TE solution, add 100 μl of 2M sodium chloride solution and 3 ml of ethanol, leave at −20 ° C. for 3 hours, and collect the formed precipitate by centrifugation. Then, it was dissolved in 10 μl of TE to purify liver-derived RNA.

Step 3 (Preparation of double-stranded cDNA using cDNA synthesis kit) A commercially available cDNA synthesis kit [manufactured by Amersham (UK)] was used for preparation. That is, 0.75 μg of the purified RNA obtained in step 1 was added to the reagents and 2 attached to the kit.
μl of random hexanucleotide primer, 2 μl of reverse transcriptase solution were added, and the total volume was adjusted to 20 μl with distilled water.
After incubation at 42 ° C. for 40 minutes, the first strand of cDNA was synthesized. Then, the second strand was synthesized while cooling in cold water. That is, 37.5 μl of the second strand synthesis reaction buffer attached to the kit, 1 μl of E. coli ribonuclease H, and 6.6 μl of DNA polymerase I were added to 20 μl of the reaction solution.
Was added, and 34.9 μl of distilled water was further mixed. 12
After reacting at 60 ° C for 60 minutes and then at 22 ° C for 60 minutes, further incubate at 70 ° C for 10 minutes, return to cold water, and
Add 1 μl of 4 DNA polymerase and repeat at 37 ℃.
After incubation for 4 minutes, 4 μl of 0.25 MEDTA
(PH 8.0) was added to stop the reaction, and phenol /
Mix well with chloroform mixture and centrifuge (12,000).
(× g, 1 minute), and the aqueous layer was separated. After repeating this extraction operation once more for the aqueous layer, chloroform was added in an equal amount,
After mixing well, it was centrifuged to separate the aqueous layer, an equal amount of 4M ammonium acetate and twice the amount of ethanol were added, and the double-stranded cDNA purified at -70 ° C was precipitated. 50 μl of precipitate
Of 2M ammonium acetate, 100 μl of ethanol was added, the mixture was reprecipitated at −70 ° C., and centrifuged (12,
(000 × g, 10 minutes), the precipitate is collected and dried,
Dissolved in 20 μl TE.

Step 4 [PCR (Polymeras
e Chain Reaction) method duplex c
Preparation of DNA] Using the RNA prepared in Step 1 or Step 2 as a template, cDNA prepared by reverse transcriptase is subjected to PCR method (Saiki, RK, Gelfand, DH,
Stoffer, S.M. , Scharf, S .; J. , Hi
guchi, R .; Horn, G .; T. , Mulli
S.K. B. , And Errich, H .; A. Pri
mer-directed enzymatic am
plication of DNA with a
thermostable DNA polymer
se. Science 239 : 487-491, 1
988) and amplified and prepared. That is, 5 to 1,000 ng of the RNA prepared in Step 1 or Step 2 was added to 20 μl of a reverse transcriptase solution {50 mM Tris.HCl.
pH 8.3, 40 mM KCl, 6 mM MgC
l ₂ , 1 μM 3′-primer (794 in FIG. 2-14
Synthetic oligonucleotide consisting of 25 nucleotides from 9 to 7973), 10 mM dNTP and 0.5 unit of reverse transcriptase [New England
Bio Lab. , (US)]} at 37 ° C. for 30 minutes. Then, 80 μl of PCR reaction solution {18 mM Tris · HCl, pH 8.3, 48 mM
KCl, 1.5 mM MgCl ₂ , 0.6 μM 5′-primer (7612 to 7636 in FIG. 2-13).
Synthetic oligonucleotide consisting of 25 nucleotides up to the second) and 3'-primer as described above, 10 mM
No. dNTP and 2.5 units of Taq DNA polymerase [Perkin Elmer Cetus (USA)]} were added, and 94 ° C. for 1 minute, 50 ° C. for 2 minutes, 72
Amplified / prepared cDNA by agarose gel electrophoresis after 40 cycles of incubation for 3 minutes at ℃
Was recovered. After phenol treatment and ethanol precipitation, it was dried and dissolved in 10 μl of TE.

Step 5 (cD using lambda gt11
Preparation of NA library) Commercially available cDNA cloning kit [Amersham
Company (UK)] was used to prepare a cDNA library. To 130 ng of cDNA prepared in step 3, 2 μl of L / K buffer attached to the kit, 2 μl of EcoRI adapter, 2 μl of T4 DNA ligase and distilled water were added to make 20 μl in total volume, and after incubation at 15 ° C. for 16 to 20 hours, 0.25M 2 μl of EDTA
In addition, the reaction was stopped. Then, after passing through the size fractionation column attached to the kit to remove the EcoRI adapter not ligated to the cDNA, 700 μl of the EcoRI adapter-ligated cDNA was added to L / K buffer 8
Add 3 μl and 8 μl of T4 polynucleotide kinase and incubate at 37 ° C. for 30 minutes. Next, phenol extraction is performed twice, and 350-400μ with butanol.
Concentrate to 1 l and ethanol precipitate to obtain 5 μl TE
Dissolved in.

Next, the cloning vector lambda gt1
CDN with EcoRI adapter added to EcoRI site of 1
In order to insert A, the above-mentioned adapter-added cDNA1
μl (10 ng) to 1 μl L / K buffer, lambda g
2 μl (1 μg) of t11 arm DNA and T4 DNA
Ligase (2 μl) was added, the total amount was adjusted to 10 μl with distilled water, and the mixture was incubated at 15 ° C. for 16 to 20 hours to prepare a recombinant lambda gt11 DNA solution. In addition, in
Gigapa of a commercially available in vitro packaging kit [Stratagene (USA)] was used for performing tro packaging to obtain recombinant lambda phage.
ckII Gold solution A (10 μl) and solution B (15 μl) were added to the above recombinant lambda gt11 DNA solution (4 μl).
After incubating at 0 ° C for 2 hours, 470 µl of the attached SM buffer and 10 µl of chloroform were added, and the recombinant phage was stored at 4 ° C.

Step 6 (E. coli plasmid pUC19
CDNA cloning using Escherichia coli plasmid pUC19 (C.Ya) using a commercially available DNA ligation kit (Takara Shuzo).
Nishi-Perron, J. et al. Vieira, J .; M
Essing, Gene 33, 103, 1985) and cloned in E. coli. P in step 4
5 μl of cDNA prepared by CR method and restriction enzyme SmaI
Dephosphorylated plasmid pUC19 DN after cleavage with
To 5 μl (50 ng) of A, 40 μl of solution A and 10 μl of solution B included in the kit are added, and the mixture is incubated at 15 ° C. for 16 hours. The plasmid DNA thus obtained
E. coli strain JM109 (Messing, J., Cre
a.R. , And Seeburg, P .; H. Nuc
leic Acids Res. 9,309,198
1) calcium chloride method (Mandel, M. and
A. Higa, J .; Mol. Biol. , 53, 15
4, 1970) to obtain transformed Escherichia coli having a plasmid incorporating cDNA.

Step 7 (N from cDNA library
Screening for Clone Carrying ANBV Gene E. coli Y1090 strain (Richard A. Young
and Ronald W. Davis, Science
CE, 222, 778, 1983) in 50 ml of LBM
Cultured in a medium (1% tryptone, 0.5% yeast extract, 1% sodium chloride, 50 μg / ml ampicillin, 0.4% maltose) at 37 ° C., and 15 ml of 10 mM sulfuric acid ice-cooled in a logarithmic growth phase. Suspended in magnesium.
The phage solution obtained in step 5 was treated with SM buffer (0.1 M sodium chloride, 8 mM magnesium sulfate,
50 mM Tris.HCl pH 7.5, 0.01%
Gelatin solution) and add 0.1m to the diluted phage solution.
1 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 was added, and L-agar medium (1% tryptone, 0.5% yeast extract, 1% sodium chloride, 1.
5% agar, 100 μg / ml ampicillin, pH 7.
Plate 0) and incubate at 42 ° C for 3 hours. Then, 10 mM IPTG (Isopropy
l β-D-thiogalactopyranosi
Soak the de) into a nitrocellulose filter, let it dry, and bring it into close contact with the plate.
Incubated for 3 hours at ° C. Filter TBS
The plate was washed three times with a buffer, immersed in a 2% bovine serum albumin solution, and incubated at room temperature for 1 hour. Commercially available immunoscreening kit [Amersham (UK)
1/20 volume of the E. coli lysate attached to the product was incubated at room temperature for 30 minutes, and NANB was diluted 50 times with TBS buffer containing 0.2% bovine serum albumin.
Soak the above filter in hepatitis patient serum and
Reacted for hours.

The filter was washed 4 times with TBS buffer containing 0.05% Tween 20, and 100% of peroxidase-labeled anti-human IgG [manufactured by Cappel (West Germany)] was used.
Immerse in 0-fold diluted antibody solution for 1 hour, then add Tween-TBS
Wash with liquid and add 0.4m to 50ml TBS buffer.
l DAB (3,3'-diaminobenzidi
ne tetrahydrochloride) and 30
The solution was immersed in a solution containing 15% hydrogen peroxide solution, incubated at room temperature for 5 to 30 minutes to develop color, and the filter was washed with water to stop the reaction.

By the above operation, the obtained plaques were purified and 9 positive clones were isolated. These clones were cloned into BK102, BK103, BK105,
BK106, BK108, BK109, BK110, B
It was named K111 and BK112. None of these clones reacted with normal human serum, but specifically with NANB hepatitis patient serum (Table 1).

Table 1 Reactivity of NANBV Hepatitis Patient Serum and Recombinant Lambda gt11 Phage Clones Normal Human Clone Hemolysis NANB Hepatitis Patient Serum BK102 0/10 * 10/11 BK103 0/10 9/11 BK105 0/10 11/11 BK106 0/10 11/11 BK108 0/10 9/11 BK109 0/10 9/11 BK110 0/10 9/11 BK111 0/10 9/11 BK112 0/10 10/11 *: Number of positives / Number of samples

Step 8 (Determination of nucleotide sequence of the obtained clone) Recombinant phage D of clones BK102 to BK112
NA was recovered and digested with restriction enzyme EcoRI, then NANB
The V cDNA fragment was isolated and E of plasmid pUC19 was isolated.
Incorporated into the coRI site. Escherichia coli JM109 strain was transformed with the thus obtained plasmid as in step 6. Plasmid DNA from these transformed E. coli
7-DEAZA sequence kit (manufactured by Takara Shuzo Co., Ltd., Mizusawa, S., Nishimura,
S. and Seela, F .; , Nucleic Ac
ids Res. , 14, 1319, 1986) to determine the nucleotide sequence of NANBV cDNA. The interrelationship of each cDNA clone is shown in FIG.

Step 9 (by Genewalking,
Cloning of NANBV cDNA clone from cDNA library) cDNA fragments of clones BK102, BK106 and clone BK112 obtained in step 8 were prepared with ³² P-dCTP labeled probes, and hybridized from the cDNA library of lambda gt11 obtained in step 5. A phage clone carrying NANBV cDNA was obtained by. That is, clone BK1 obtained in step 8
02, BK106 and BK112 transformed from E. coli by the alkaline method (T. Maniatis, EF Frit.
sch, and J. Sambrook: Isolat
ion of Bacteriophage λ an
dPlasmid DNA: "Molecular C
longing "ColdSpring Harbor
Lab. , Pp75-96. ) Was used to prepare plasmid DNA. The plasmid DNA of the clone BK102 was subjected to agarose gel electrophoresis of a 0.7 Kb fragment on the 5'end side generated by digestion with restriction enzymes NcoI and HincII,
Recovered. The plasmid DNAs of clones BK106 and BK112 were digested with the restriction enzyme NcoI to give a 1.1 Kb DNA fragment from clone BK106.
From clone BK112, a 0.7 Kb fragment on the 3'end side was recovered. The DNA fragment of 25 ng to 1 μg was prepared by using a commercially available DNA labeling kit (Nippon Gene Co., Ltd.) [α- ³² P] dCTP [3000 Ci / mm.
ol, manufactured by Amersham (UK)], and a probe for hybridization was prepared by incubating at 37 ° C. for 3 to 5 hours.

Next, the phages of the cDNA library obtained in step 5 were L-ligated as described in step 7.
After incubation in agar medium at 42 ° C. for 3 hours and then at 37 ° C. for 3 hours, the mixture is cooled, put on a nitrocellulose filter and left for 30 to 60 seconds to adsorb the phage to the filter. Alkali denaturation (0.5N sodium hydroxide-1.5M sodium chloride solution for 1-5 minutes),
Neutralization (0.5 M Tris.HCl pH 8.0-1.
After treatment with 5M sodium chloride solution for 1 to 5 minutes), 2 x SSC
It is washed with (0.3 M sodium chloride-0.03 M sodium citrate solution), air-dried, and baked at 80 ° C. for 2 hours. Then filter the hybridization solution (5
0% formamide, 5 × SSC, 5 × Denhart
Solution, 50 mM phosphate citrate buffer pH 6.5, 1
4 with 00 μg / ml salmon sperm DNA, 0.1% SDS)
After incubating at 2 ° C. for 6 hours, about 4 × above
Immerse again in 300 ml of the above hybridization solution containing 1 ml of 10 ⁸ cpm / ml probe at 42 ° C.
Incubate for 16-20 hours and wash (2 x SS
C-0.1% SDS solution 4 times, 0.1 × SSC-0.1
% SDS solution twice), followed by drying and autoradiography to isolate clones showing positive hybridization. As a result, 27 clones that react with the probe derived from BK102, 14 clones that react with the probe derived from BK106, and 13 clones that react with the probe derived from BK112 could be obtained. Each clone was sequentially named as BK114 to BK169. Furthermore, when the nucleotide sequence was determined as described in Reference Example 8 and the mapping of each clone was performed, it was mapped to a size of about 9.5 Kb, which is considered to be almost the entire length of the NANBV genome (Fig. 1-2). Of these, a 0.55 Kb fragment on the 5'side from clone BK157 located on the 5'end side was recovered after digestion with the restriction enzyme KpnI, and 0.55 Kb on the 3'end side from clone BK116 located on the 3'most end side. Fragment was recovered after digestion with the restriction enzymes HpaI and EcoRI, and a probe labeled with ³² P was prepared as described above.
The phages of the cDNA library obtained in Step 1 were plaque-hybridized, and three new clones were isolated with a probe derived from BK157.
70, BK171 and BK172.

Step 10 (Analysis of base sequence of cDNA) FIGS. 2-1 to 2-16 are a summary of the base sequences of the clones obtained in Steps 8 and 9.
As a result, the cloned NANBV genomic cDNA
NA consists of 9416 bases in total, within which 9030
There was an open reading frame consisting of bases (translational reading frame), 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 (H. Sumyoshi, C. Mo.
ri, I. Fuke et al. , Complete
Nucleotide Sequence of the
e Japanese Encephalitis Vi
rus Genome RNA. Virology,
161 , 497-510, 1987).
The clone BK157 covers the nucleotide numbers 1 to 1962 from FIGS. 2-1 to 2-16, BK172 from the 5th to 366th, and BK153 from the 338th to 1st.
Up to 802, BK138 is 1755 to 512
Up to the 4th, BK129 covers the 4104th to 6973rd, BK108 covers the 6886th to 8344th, and BK166 covers the 8082th to 9416th. In addition, these clones are
It is a part of a group of clones constituting the NBV gene cDNA / BK series, and is Escherichia coli.
BK108 (Ministry of Fine Arts, Article 2971), BK129
(Ministry of Fine Arts, Article 2972), BK138 (Ministry of Fine Arts, Article 2973), BK153 (Ministry of Fine Arts, Article 2974)
No.), BK157 (Microtechnical Laboratory Article No. 3243), BK1
It was preserve | saved as 66 (Microtechnology Research Article No. 2975) and BK172 (Microtechnology Research Article No. 2976).

Reference Example 1 [Production of Various Antigens Associated with Antibody Responses Associated with NANBV Infection (hereinafter referred to as NANBV-Related Antigens) by E. coli] BK106, BK in Step 8 of Example 1 (Part I)
The plasmid DNA in which the cDNA of each of 111, BK112 and BK147 obtained in step 9 of Example 1 (Part I) was inserted was recovered by the alkaline method. Then, the DNA of BK106 was digested with restriction enzymes EcoRI and Cla.
Of the 0.34 Kb DNA fragment digested with I and recovered.
5 μg of T4 DNA polymerase reaction solution (67 mM T
ris HCl pH 8.8, 6.7 mM magnesium chloride, 16.6 mM ammonium sulfate, 10 mM 2-mercaptoethanol, 6.7 μM EDTA, 0.02%
Bovine serum albumin, 0.3 mM dNTP and 2-5
Unit T4 DNA polymerase) at 37 ° C. at 60
Minutes, and both ends were blunted. BK10
The DNA of 2 was digested with the restriction enzyme BamHI, and 0.5 μg of the recovered 0.7 Kb DNA fragment was treated with T in the same manner as above.
The ends were made blunt using 4 DNA polymerase. BK
The 147 DNA was digested with the restriction enzyme Sau3AI, and 0.5 μg of the recovered 1 Kb DNA fragment was blunt-ended in the same manner as above. The BK111 DNA was digested with the restriction enzyme EcoRI, and 0.5 μg of the recovered 1 Kb DNA fragment was blunt-ended in the same manner as above. Next, the expression vector pKK233-2 (Amann, E. an.
d J. Brosius. ATG vector fo
r regulated high-level ex
compression of cloned genes
in Escherichia coli. Gene,
40 , 183, 1985) to the restriction enzyme Hind
After digestion with III, 2 μg of DNA was incubated in S1 nuclease reaction solution (0.3 M sodium chloride, 50 mM sodium acetate pH 4.5, 1 mM zinc sulfate, 100 to 200 units of S1 nuclease) at 37 ° C. for 20 minutes. , 0.12M EDTA and 1M Tris HC
The reaction was stopped by adding 1/10 volume of pH 9.0 solution, phenol extraction was performed, and vector DNA having blunt ends was precipitated with ethanol and recovered. The DNA of the vector pKK233-2 was digested with the restriction enzyme PstI, purified by phenol extraction and alcohol precipitation, and then 2 μg was obtained by the above T4 DNA polymerase reaction.
The ends cleaved by PstI of the vector DNA of 1. were blunted. The clones BK106 and BK thus obtained
The 111-derived DNA fragment was blunt-ended after vector cleavage with HindIII, and the clones BK102 and BK147-derived DNA fragments were Ps and Ps, respectively.
After cleavage with tI, mix with vector DNA with blunt ends (0.5 μg of insert cDNA fragment and vector DNA
Each) 2 μl of 10 × ligation solution (500 mM
Tris.HCl pH 7.5, 100 mM magnesium chloride, 100 mM DTT, 10 mM ATP),
300 to 400 units of T4 DNA ligase and distilled water were added to make a total volume of 20 μl, and the mixture was incubated at 14 ° C. for 12 to 18 hours.
It was named E-06, pE-11, pB-02, pS-09. Next, E. coli JM109 strain was transformed with the above plasmid DNA as described in step 6 of Example 1 (Part I) to obtain transformed E. coli. The Escherichia coli thus obtained was LB medium (1% W / V tryptone, 0.
Using 5% W / V yeast extract, 1% W / V sodium chloride, pH 7.5), the cells are cultivated at 37 ° C., 1 mM of IPTG is added in the logarithmic growth phase, and further cultivated for 3 hours. The bacterial cells were collected by centrifugation (10,000 × g, 15 minutes), and 50 mM Tris · HCl pH 8.0 was added.
And sonicate (20 KHz, 600 W, 5 minutes), and then centrifuge (10,000 xg, 15 minutes) to separate into a supernatant and a precipitate.
0% V / V glycerol, 0.1M Tris / HC
l pH 6.8, 2% W / V SDS, 2% V / V
2-mercaptoethanol, 0.02% BPB), and heated at 100 ° C. for 3 minutes, and then 0.1% SDS-
After 7.5% polyacrylamide gel electrophoresis, the protein was trans-blotted [BIO-RAD (USA)]
Transfer to a nitrocellulose filter at. Then
Soak the filter in 3% gelatin solution and leave it for 60 minutes, then
After incubating with NANB hepatitis patient serum diluted 1:00 (room temperature, 2 to 3 hours) and washing with water, TTBS solution (0.
Wash with 02M Tris.HCl pH 7.5, 0.5M sodium chloride, 0.05% V / V Tween 20). Next, it is immersed in a 2000-fold diluted peroxidase-labeled anti-human IgG antibody solution and incubated at room temperature for 90 minutes. After washing with distilled water, wash with TTBS. Next, as described in Step 7 of Example 1 (Part I), the color former DAB and the substrate 30% hydrogen peroxide solution are immersed in the buffer for 5 to 30 minutes and washed with water to stop the reaction.

As a result, as shown in Table 2, which type of
The antigen produced by rasmid is also the serum of NANB hepatitis patients.
CD that reacts specifically and is inserted into these plasmids
The protein produced by NA has been shown to be clinically important.
It was Table 2 Reactivity of proteins produced by various plasmids and serum of NANB hepatitis patients by Western blotting cDNA NANB hepatitis healthy subjects Origin of plasmid Extract Serum Serum Serum pCE-066 BK106 S ± − P + − pE-11-89 BK111 S ± − P + − pB-02-10 BK102 S + − P − − pS-09-7 BK109 S ± − P + − pKK233-3- − S--P − − S: Centrifugal supernatant P: Centrifugal sediment +: Positive ±: Weakly positive −: Negative

Reference Example 2 (Purification of NANBV-related antigen produced by Escherichia coli and reactivity with serum of liver disease patients) cDNA inserted in expression vector
The usefulness of the proteins produced by A is to purify these proteins,
It can also be shown by using it as an antigen for ELISA or radioimmunoassay. That is, the cell disruption solution of the transformed Escherichia coli obtained in Reference Example 1 was centrifuged (10,000 ×
(g, 15 minutes). For example,
The precipitate obtained from the transformant JM109 / pCE066 was treated with 100 mM Tris.HCl pH 8.0-0.1.
% Triton X-100 solution, followed by ultrasonic treatment (20 KHz, 600 W, 1 minute) and centrifugation (2,0).
(00 × g, 15 minutes), and the precipitate was added to 100 mM Tri
After resuspending in s.HCl pH 8.0-6M urea, ultrasonic treatment is performed. The supernatant obtained by centrifugation is 10 mM
Phosphate buffer solution, dialyzed against pH 7.5-6M urea, and 20 ml of the antigen solution was equilibrated with the same buffer solution for high-performance liquid chromatography (HPLC) hydroxyapatite column (2).
(1.5 × 250 mm), a 0-2 M linear concentration gradient of sodium chloride is drawn out in the equilibration buffer, and the fractions containing the antigen are pooled. Then, 50 mM carbonate buffer pH 9.6-0.05% sodium dodecyl sulfate (S
It was dialyzed against DS). In addition, the transformant JM109 /
Centrifugal supernatant (10,000) of the disrupted cell suspension of pB-02-10
(0 × g, 15 minutes) was treated with 35% saturated ammonium sulfate, and the obtained precipitate was treated with 50 mM Tris · HCl pH 8.5-10.
It is dissolved in 0 mM 2-mercaptoethanol and dialyzed against the same buffer. Then HPL equilibrated with the same buffer
DEAE-cellulose column for C (22.0 x 200 m
100 ml of dialyzed sample was added to m), and 50 mM T
Fractions containing antigen are pooled by elution with a 0-2M linear gradient of sodium chloride in ris.HCl pH 8.5-100 mM 2-mercaptoethanol. The antigen solution is 10 mM phosphate buffer pH 6.8-100 mM 2-
After being dialyzed against mercaptoethanol and adsorbed on a hydroxyapatite column for HPLC equilibrated with the same buffer solution, a 10 to 400 mM linear concentration gradient elution of phosphoric acid was performed, and the fractions containing the antigen were pooled and 50 mM carbonate buffer was added. Liquid p
It was dialyzed against H9.6-0.05% SDS. Furthermore, centrifugation precipitation of the disrupted cell of the transformant JM109 / pE-11-89 was suspended in 10 mM phosphate buffer pH 5.5, sonicated for 1 minute, and centrifuged (21,000).
Xg for 15 minutes). The obtained precipitate is 100 mM carbonate buffer pH 10.5-500 mM sodium chloride-1.
The suspension is suspended in 0 mM EDTA solution, sonicated again for 1 minute, and the centrifuged supernatant is dialyzed against 30 mM phosphate buffer-6 M urea. Then, a CM-cellulose column for HPLC (22 × 2) equilibrated with the same buffer as that used for dialysis was used.
(00 mm) adsorbing 20 ml of the sample, and elution with a 0 to 1.5 M linear concentration gradient of sodium chloride in the same buffer, pooling the fractions containing the antigen, and adding 50 mM carbonate buffer
The antigen solution was prepared by dialysis against pH 9.6-0.05% SDS.

The antigen prepared as described above was assayed by ELISA.
It can be used as an antigen for clinical diagnosis of NANB hepatitis virus infection. That is, the purified antigen was added to a protein concentration of 1 μg
/ Ml, 100 μl of the solution is added to each well of an ELISA microplate Imron 600 [IMMULON, Greiner (West Germany)], and the plate is allowed to stand at 4 ° C. overnight. Each well contains PBS-T buffer (10 mM
Phosphate buffer pH 7.2-0.8% sodium chloride-
Wash well with 0.05% Tween-20) 3 times, and
100 μl / well of test serum diluted with BS-T is added and reacted at 37 ° C. for 1 hour. After washing three times with PBS-T, peroxidase-labeled anti-human IgG antibody [Cap
Pel company (West Germany)] PBS containing 10% fetal bovine serum
100 µl diluted 8,000 times with -T buffer
Add to each well one by one, react at 37 ℃ for 1 hour, then again,
After washing 4 times with PBS-T, the substrate color developer solution (0.5 μg of o-in 9 ml of 0.05 M citrate-phosphate buffer) was used.
100 μl of 1 ml of phenylenediamine and 20 μl of hydrogen peroxide solution are added to each well, and the plate is left at room temperature for 60 minutes while protected from light. Add 75 μl of 4N sulfuric acid solution to each well and measure the absorbance at 490 nm.
As shown in Table 3, all the transformant-derived antigens specifically reacted with the serum of NANB hepatitis patients, demonstrating the clinical diagnostic usefulness of the antigens produced by these transformants.

Table 3 Origin of Reactive Antigens by ELISA of Antigens Purified from Various Transformed E. coli and NANB Hepatitis Patient SerumPost-transfusion hepatitis patient serum Healthy person(Transformed E. coli) Acute chronic Cirrhosis Liver cancer Serum JM109 / pCE-066 2/3 * 7/8 3/4 3/3 0/10 JM109 / pB-02-10 2/3 8/8 4/4 3/3 0/10 JM109 / pE-11-89 2/3 8/8 2/4 3/3 0/10 *: Number of positives / Number of samples

The results shown in Table 3 can also be obtained by radioimmunoassay using the above antigens.
That is, 0.2 ml of the above purified antigen solution having a concentration of 1 μg / ml
A 1 / 4-inch diameter polystyrene ball [made by Pazel (West Germany)] was added to each of them, and the mixture was allowed to stand overnight at 4 ° C. Then, styrene balls were washed 5 times with PBS-T used in the above ELISA, and the test serum was diluted with PBS-T at 1:20.
200 μl of each diluted 1: 2500 times from the above was added and reacted at 37 ° C. for 60 minutes. 5 with PBS-T
After washing twice, 200 μl of ¹²⁵ I-labeled anti-human IgG antibody
After adding each one and making it react at 37 degreeC for 1 hour, PBS-T
After washing 5 times with, the cpm of ¹²⁵ I bound to styrene balls was measured, and the same results as shown in Reference Table 3 were obtained. The purified antigen NAN obtained in this example was
The clinical usefulness of hepatitis B virus infection diagnosis became clear.

Reference Example 3 [PCR (Polymerase Chain Rea
Detection of NANBV nucleic acid by the method)] To prevent the development of NANB hepatitis after transfusion, it is important to test the presence or absence of infection of NANBV in blood used for transfusion, and for the diagnosis of liver disease, NANBV of liver tissue
Although it is clinically extremely important to examine the presence or absence of infection, the NADV cD obtained by the present invention is
Prepare a primer for PCR from the base sequence of NA,
NBV nucleic acids can be detected. That is, RNA is purified from 1 ml of various sera from patients and healthy individuals as described in Step 1 of Example 1 (Part I). Similarly, as described in step 2 of Example 1 (Part I),
RNA is prepared from liver cells. Then, PCR was performed as described in step 4 of Example 1 (Part I) to obtain cDNA after electrophoresis. The NANBV cDNA clone BK10 was identified by Southern hybridization according to a conventional method to determine whether the amplified cDNA was derived from NANBV.
It was carried out using a ³² P-labeled probe prepared from cDNA derived from 8. As shown in Table 4, the primers prepared from the nucleotide sequence of NANBV cDNA obtained by the present invention and the cloned NANBV cDNA were cloned.
NANB in serum by using the above fragment as a probe
Detection of V nucleic acids can be performed to diagnose serum NANBV contamination.

Table 4 Detection of NANBV Nucleic Acid by PCR Method Assay Antibody to NANBV PCR Chronic hepatitis patient serum NANB type 1 + + 2 + + HBV carrier 1 − − 2 − − healthy person 1 − − 2 − − resection liver of NANB type liver cancer −1 + cancer part + non-cancer part + resection liver of NANB type liver cancer -2 + cancer part + non- cancer part +

Example 1 (Part II) Step 1 (Construction of a plasmid for expressing all genes by NANBV Escherichia coli) BK shown in FIGS. 1-1 and 1-2.
112, BK146, BK147, BK157 and B
The cDNA was recovered from K166 to construct a plasmid for expressing all the genes of NANBV in Escherichia coli. That is, the plasmid DNA of clone BK157 was digested with BamHI, the 1.3 kb fragment was recovered from an agarose gel, and inserted and ligated into the BamHI site of plasmid pUC19 (Takara Shuzo) to prepare plasmid pBam157. The plasmid pBam157 is digested with XbaI and NcoI to recover a DNA fragment of about 3.9 kb. Next, Xb
Bacteriophage T7 RNA polymerase promoter region (TA consisting of aI linker sequence and 20 base pairs)
ATACGACTCACTATAGG) and the sequence from the 1st to 73rd of the nucleotide sequence shown in FIG.
An oligonucleotide consisting of 4 ends deleted) was synthesized and ligated with the 3.9 kb DNA fragment to prepare plasmid pDM-16. Then, pDM-16 was added to C
After digestion with laI and EcoRI, a DNA fragment of about 3.5 kb was recovered, and BK146 DNA was digested with ClaI and EcoR.
Ligation with the 4.1 kb DNA fragment obtained by digestion with I gave plasmid pDM-9. Then the plasmid pDM
-9 was digested with SacII to recover a 2.7 kb DNA fragment and a 4.9 kb DNA fragment. 4.9k
The DNA of b was ligated to the SacII site with T4 DNA ligase and then digested with BamHI and EcoRI to obtain about 7.5.
The kb DNA fragment is recovered. On the other hand, after digesting a DNA fragment of about 2 kb from BK147 with BamHI and EcoRI,
It was recovered and ligated with the above 7.5 kb DNA fragment to construct plasmid pBE147. pBE147 is Sac
2.7k recovered from pDM-9 above after digestion with II
The DNA fragment of b was inserted and ligated to obtain the plasmid pDM-B.
Got 3. XbaI and Ec from plasmid pDM-B3
After digestion with oRI, a 6.7 kb DNA fragment was recovered.
Furthermore, after digesting BamHI from BK166, a 1.3 kb DNA fragment was recovered, and a plasmid pBam166 was constructed by inserting and ligating into the BamHI site of pUC19.
Digestion with eI and HindIII recovers a 2.8 kb DNA fragment. Also, from BK112, EcoRI and Nd
A DNA fragment of about 1.6 kb was recovered by digestion with eI, and pUC19 was further digested with EcoRI and HindIII,
A DNA fragment of about 2.6 kb was recovered. 3 types of D above
Mix NA fragments and EcoR with T4 DNA ligase
A plasmid pEN112 in which the I, NdeI and HindIII sites were ligated was constructed. E from pEN112
After digestion with coRI and XbaI, a 2.7 kb fragment was recovered, and the 6.7 kb fragment obtained by digestion with EcoRI and XbaI of pDM-B3 was ligated.
Plasmids pDM-22 and p inserted and ligated into the aI site
DM-18 was obtained. pDM-18 can be transformed into animal cells and the like with plasmid DNA or RNA produced therefrom by an in vitro transcription kit (Boehringer Mannheim Yamanouchi) to produce a NANBV antigen production system. The plasmid pDM-22 in which the cDNA was inserted in the opposite direction to pDM-18 was digested with HindIII and ClaI to recover a DNA fragment of about 9 kb. On the other hand, BK
106 was digested with BamHI, a DNA fragment of about 1.0 kb was recovered, and a plasmid pBam106 was inserted and ligated into the BamHI site of pUC19 to prepare a plasmid pBam106. Blunted the ends with XbaI
After digestion with, a fragment of about 3.6 kb was recovered. This fragment was downstream of the XbaI linker from 333 to 37 in FIG.
The synthetic oligonucleotides in which the base sequences up to the second were ligated were ligated to obtain a plasmid pXb106. This plasmid pXb106 was digested with HindIII and ClaI, and then a 0.4 kb DNA fragment was recovered and ligated with the approximately 9 kb DNA fragment recovered from the above-mentioned plasmid pDM-22 to obtain plasmid pORF-24. Finally, after digesting the plasmid pORF-24 with XbaI, about 9.
A 0 kb DNA fragment was recovered and an expression vector (F. Wi.
lilm Studio B. A. Moff
att, J.M. Mol. Biol, 189 , 113, 19
86) and constructed the expression plasmid pJF-22.

Step 2 (Production and Culture of Recombinant Escherichia coli) Using the expression plasmid pJF-22 constructed in Step 1, E. coli JM109 (DE3) strain [manufactured by Promega (USA)] was subjected to the calcium chloride method (Journal o.
f Molecular Biology, 53 , 15
4, 1970) and recombinant JM109 (D
E3) / pJF-22 was obtained.

Recombinant E. coli JM109 (DE3) / pJ
As described in Reference Example 1, F-22 was cultivated in LB medium, added with 0.5 mM IPTG, and further cultivated for 3 hours. Then, the cells were collected and 2% SDS-2% 2-mercapto was added. After heating in ethanol-containing buffer at 100 ° C for 3 minutes,
Electrophoresis was performed on a gel composed of 0.1% (w / v) SDS-1.25% (w / v) polyacrylamide, and the proteins separated on the gel were subjected to nitro by a trans-blot apparatus (manufactured by Japan Eido Co.). After blotting on a cellulose membrane, the protein produced by Western blotting was analyzed. The specific antiserum used in the western blot was obtained by purifying the NANBV-related antigen prepared in Reference Example 1 and immunizing a guinea pig. As a result of this analysis, it was shown that the protein produced by recombinant JM109 (DE3) / pJF-22 reacts with all antisera (No. 5).
table). Table 5 Reactivity of recombinant Escherichia coli JM109 (DE3) / pJF-22 production protein with NAN BV-related antibody Serum of NANB hepatitis patient serum Guinea pig antiserum microbial cell extract pool serum acute chronic anti-core anti-NS3 anti- NS5 JM109 (DE3) / pJF22 + * + + + + + JM109 (DE3) - - - - - - * Western blotting with reactive recombinants to 5 'end of the genome co - core antigen that has been de From the nonstructural protein NS5 encoded at the 3'end
It was shown to express all the genes up to. Such a recombinant not only provides a very useful raw material for a diagnostic agent for NANBV infection, but also a raw material for a vaccine.

Step 3 (NANBV whole gene cDNA
Production of NANBV Particles by Expression in a Animal Cell) The plasmid pORF-24 obtained in Step 1 was transformed with XbaI.
After digestion with, this was subjected to low melting point agarose gel electrophoresis to recover a DNA fragment of about 9 kb. Next, NheI
Plasmid cleaved with pMAM-neo (Clonte
The expression plasmid pMAM-neo10 was constructed by inserting and ligating into ch company (purchased from USA) (FIG. 4). The plasmid is then transferred to the calcium phosphate method ("Molecular Cloning", 16.
33-16.39, Cold Spring Harb
or Laboratory, 1989), human liver-derived cells, Chang Liver (ATCC).
CCL13) and chimpanzee liver-derived cells (purchased from Dainippon Pharmaceutical Co., Ltd.).
The cells into which the plasmid pMAM-neo10 was introduced were transformed into the antibiotic aminoglycoside G418 resistance, and G4
Since colonies were formed in the presence of 18 600 μg / ml, transformants were selected and cloned using this as an index. Transformed cell clones HL-A1 and HL-A2 obtained from human-derived cells, and transformed cell clone CL-B1 obtained from chimpanzee liver-derived cells
1. Cover glass placed in a petri dish at 37 ° C. for 4 days in Eagle MEM medium containing 5% (v / v) fetal bovine serum in the same manner as described in Reference Example 4 below with CL-B14. It was cultured on. The protein produced by cell culture was assayed for the presence or absence of various NANBV antigens by the conventional indirect fluorescent antibody method using the specific antiserum described in Step 2. As a result, the protein produced in the G418-resistant cell clone reacted with all antisera of guinea pigs immunized with the NANBV-related antigen prepared in Reference Example 1 (Table 6).

Table 6 Detection of NANBV-related Antigens Produced by Recombinant Human and Chimpanzee-Derived Hepatocytes by Fluorescent Antibody Method Hepatitis Patients with NANB Hepatitis Serum Guinea Pig Antiserum Cell Pool Serum Acute Chronic Anti-Core Anti-NS3 Anti-NS5 Human Liver-derived HL-A1 ++ + ++ + HL-A2 + ++ + + + + Normal Chang Liver − − − − − − Chimpanzee liver-derived CL-B11 + + + + + + CL-B14 + + + + + Normal Chimp Liver − − − − − − This means that the entire region of NANBV from C protein to NS5 was expressed.

Step 4 (Human or chimpanzee hepatocyte-derived transformed cell clones HL-A1 and CL-B
Sucrose density gradient centrifugation of NANBV-related antigen produced by 11) Clones HL-A1 and CL-B11 were placed in a carbon dioxide incubator at 37 ° C., 4 ° C. in 5 petri dishes for tissue culture each having a diameter of 9 cm, as described in step 3. Cultured for a day. Remove cells with a rubber policeman, pool with culture medium, sonicate (20 kHz, 200 W, 2 minutes), and centrifuge (500
The supernatant obtained by 0xg, 15 minutes, 4 ° C) was further centrifuged (48.0).
(00 × g, 14 hours, 4 ° C.), and the precipitate was mixed with 1 ml of M / 7.
Float in 5 PBS and sonicate for 2 minutes
0 w / v% to 60 w / v% sucrose density gradient centrifugation (16
50,000 × g, 15 hours, 4 ° C.) and then fractionated. Each fraction was subjected to SDS-polyacrylamide gel electrophoresis according to the method described in Step 2 and then subjected to Western blotting to detect the core antigen and the envelope antigen. As a result, both antigens were detected at a sucrose density of around 40 w / v% to 50 w / v%, and it was shown that NANBV particles were obtained.

Example 2 Step 1 (Construction of plasmid for expression of NANBV whole gene cDNA by yeast and production of recombinant yeast) The plasmid pORF24 obtained in Example 1 was digested with XbaI to prepare the plasmid of Example 1 (Part II). Similar to step 1, a NANBV cDNA fragment of about 9 kb was recovered. This cd
0.5 μg of NA fragment was added to T4 DNA polymerase reaction solution (67 mM Tris.HCl pH 8.8, 6.7 m).
M magnesium chloride, 16.6 mM ammonium sulfate,
10 mM 2-ME, 6.7 mM EDTA-2Na
0.02% w / v% bovine albumin, 0.3 mM dN
2 to 5 units of T4 DNA polymerase (manufactured by Takara Shuzo Co., Ltd.) was added, and the mixture was incubated at 37 ° C. for 60 minutes to make both ends blunt ends. Next, the XhoI linker (CCTCGAGG) is ligated with T4 DNA ligase. That is, 0.3 μg of DNA was added to 21 μl of 10 ×
Ligation solution (500 mM Tris-HCl p
H7.5, 100 mM magnesium chloride, 100 mM
Dissolve in DTT, 10 mM ATP), 300-400
A unit of T4 DNA ligase (manufactured by Takara Shuzo Co., Ltd.) and distilled water were added to make 210 μl in total volume, and the mixture was incubated at 14 ° C. for 12 to 18 hours. The thus-prepared cDNA fragment was inserted and ligated into the XhoI site of the yeast expression vector YEp133PCT (US Pat. No. 4,810,492) to obtain the expression plasmid pYHC5 (FIG. 5).
With this expression plasmid pYHC5, the yeast S. ce
revisiae (ATCC No. 44772) was subjected to an alkali cation method (Ito, H. et al. J. Ba.
cteriol. , 153: 163-168 198.
Transformation was performed in 3) to obtain recombinant yeast YHC5-1.
This recombinant plant is designed to produce NANBV-related antigens by activating the yeast inhibitory acid phosphatase gene promoter when the phosphate ion in the medium is deficient and causing transcription of NANBV cDNA linked downstream, Has been built.

Step 2 (Production of NANBV-related antigen by yeast and its properties) The recombinant yeast YHC5-1 obtained in Step 1 was prepared by using a bulk holder medium which is a complete synthetic medium containing 1.5 g / l potassium phosphate monobasic. (Burkholder, PR et a.
l. , Am. J. Botany 30,206-21
1, 1943) to uracil, L-tryptophan, L-
Histidine was inoculated into 100 ml of a medium prepared so as to contain 20 μg / ml each, and the mixture was shake-cultured at 30 ° C. for 24 hours. Then 1000 ml of the above fresh medium containing 1.5 g / l potassium chloride instead of potassium monophosphate is inoculated with the above yeast washed with physiological saline. Then, the cells were cultured at 30 ° C. for 24 hours to collect the cells, and
5 PBS, glass beads (diameter 0.45-0.
55mm) and add bead beater (BiospecP)
The cells are crushed by products (US)
A supernatant obtained by centrifugation (10,000 × g, 10 minutes, 4 ° C.) was obtained. The supernatant thus obtained was subjected to SDS-polyacrylamide gel electrophoresis by Western blotting according to the method described in Step 2 of Example 1 (Part II), and then NANB was analyzed.
The presence or absence of V-related antigen production was examined. As a result, the extract of recombinant yeast YHC5-1 reacted with all of the antibodies against the core antigen, envelope antigen, NS3 protein and NS5 protein. This means that all regions from core antigen to NS5 were expressed (Table 7).

Table 7 Reactivity of recombinant yeast YHC5-1 production protein with NANBV-related antibody Serum NANB hepatitis patient serum Guinea pig antiserum microbial cell extract pool serum acute chronic anti-core anti-NS3 anti-NS5 YHC5- 1 ++++++++ Normal S. cerevisiae − − − − − * Reactivity by Western blotting: Also, sucrose density gradient centrifugation was performed in the same manner as in step 4 of Example 1 (Part II). However, it was shown that both the core antigen and the envelope antigen were detected in the fraction having a sucrose concentration of 40 w / v% to 50 w / v%, and NANBV particles were obtained.

Reference Example 4 (Culture of vaccinia virus WR strain) Vaccinia virus was cultured according to a conventional method. That is, mouse-derived thymidine kinase (TK) -deficient cell line LM cultured in a monolayer
(TK) (Original ATCC strain number CCL-1.3, Dainippon Pharmaceutical Co., Ltd.), monkey kidney-derived Vero cells or adult liver-derived cells Chang River (original ATCC strain number C)
CL-13, Dainippon Pharmaceutical Co., Ltd., etc.) were inoculated with 0.5 ml of vaccinia virus into cells cultured in a 6 cm dish and left at 37 ° C. for 1 to 2 hours to remove the virus solution. Then, 5 ml of Eagle MEM medium (Nissui Pharmaceutical Co., Ltd.) supplemented with 5 v / v% fetal bovine serum was added to the virus culture solution, and 3
After culturing at 7 ° C for 24 to 48 hours, and when sufficient cell degeneration has occurred, collect the virus culture solution or infected cells and
After floating in EM, ultrasonic treatment (20kHz, 200W, 2
Minutes) and used as a virus solution.

Reference Example 5 (Infectious titer measurement of vaccinia virus by plaque method)
The plaque method was performed according to a conventional method. That is, 0.1 ml / 6 of the virus solution of Reference Example 4 diluted 10-fold with M-199 (Sigma [USA] Inc.) was added to the cell culture described in Reference Example 4.
cm dish, incubate at 37 ° C. for 2 hours and adsorb the virus to the cells, remove the inoculum and add agar-containing medium (M-199 to fetal bovine serum 3 v / v%, NaHCO ₃
0.14w / v%, agar 0.8w / v% added) 5m
1 / dish each were added. When the agar solidifies at room temperature,
After culturing at 37 ° C for 24 hours, 2.5 ml / dish of a medium prepared by adding about 0.006 w / v% of neutral red (Wako Pure Chemical Industries) to the above agar medium was overlaid, and the culture was continued at 37 ° C. The number of emerged plaques was measured to determine the infectious titer.

Example 3 Step 1 (Construction of plasmid to be inserted into vaccinia virus) Plasmid pUV1 (Falko G. Fal
kner, Sekhar Chakrabarti a
nd Bernard Moss; Nucleic A
cid Res. , 15 (17), 7192, 198
7) was digested with restriction enzyme EcoRI, 0.5 μg of the DNA recovered by phenol precipitation after ethanol extraction was dissolved in the T4 DNA polymerase reaction solution as described in Reference Example 1, and 2 to 5 units of T4 DNA polymerase (Takara Shuzo) was added. In addition, both ends were blunted by incubating at 37 ° C. for 60 minutes. On the other hand, the DNA fragment having the entire gene sequence encoding the NANBV protein was prepared as in Example 1.
Plasmid pOR as described in step 1 of (Part II)
F-24 is a restriction enzyme XbaI or XbaI and EcoR
About 9 kb or 6.4 kb of D obtained by digestion of I
The NA fragment was recovered, treated with T4 DNA polymerase in the same manner as above, and blunted at both ends to obtain the fragment. 0.5 μg of pUV1-derived DNA thus obtained and pOR
0.5 μg of F-24-derived DNA was dissolved in 21 μl of 10 × ligation solution as described in Reference Example 1, and 30
Add 0 to 400 units of T4 DNA ligase (Takara Shuzo) and distilled water to a total volume of 210 μl, and add 12 to 1 at 14 ° C.
After incubation for 8 hours, NANBV-derived cD was located at the EcoRI site downstream of the pUV1 promoter.
NA was linked. The reaction solution was phenol-extracted, the aqueous layer was precipitated with ethanol to recover DNA, and Escherichia coli JM109 strain was transformed by the transformation method of Escherichia coli with calcium chloride as described in step 6 of Example 1 (Part I). Plasmid clones pXX-49 and pXX-51 having a NANBV cDNA fragment of about 9 kb were obtained.
In addition, about 6.4 kb N lacking the NS5 region of NANBV
Plasmid pXE-39 carrying the ANBV cDNA fragment
Got The result is shown in FIG.

Step 2 (Production of Recombinant Vaccinia Virus) Vaccinia virus WR strain (ATCC VR-119) was added to MOI (Vaccinia virus WR strain) in Vero cells cultured in a tissue culture dish having a diameter of 9 cm (Falcon, USA) for 24 hours. M
multiplicity of infection)
After inoculation with 0.05, the virus was adsorbed to the cells at 37 ° C. for 2 hours, the virus solution was removed, and the cells were washed twice with MEM. Then, the plasmid D obtained in Step 1 of Example 3
NA in an amount of 1 μg and 5 μg each was measured by the calcium phosphate method (Graham, FL, van der Eb, A .;
J. : Virology, 52, 456-467, 19
73) DNA-calcium phosphate precipitation in 1)
It was prepared one by one. Add 1 ml each of this DNA / calcium phosphate precipitate to the above virus-infected cells and bring to room temperature for 30 minutes.
After leaving to stand for 15 minutes, 15 ml of each virus culture solution described in Reference Example 4 was added and incubated at 37 ° C for 3.5 hours. Next, the virus culture solution was removed, replaced with 15 ml of a fresh virus culture solution, and cultured at 37 ° C. for 48 hours. The virus suspension obtained by repeating freeze-thaw three times was inoculated into LM (TK) cells cultured in a culture dish having a diameter of 6 cm according to the method described in Reference Example 5. After virus adsorption,
5-Bromo-2'-deoxyuridine (B
UdR, Sigma) 25 μg / ml-containing agar medium (5 ml / dishes) was overlaid and cultured at 37 ° C. for 8 days.
Chloro-3-Indolyl-β-D-Gala
25 μg / m of ctoside (X-Gal, Takara Shuzo)
The agar multi-layer medium containing 1 was overlaid at 2.5 ml / dish and further cultured at 37 ° C. for 2 days. The blue plaque that appeared was collected together with the layered agar medium, and 0.5 ml of M-19 was collected.
9 and suspended the supernatant in the same manner as above, using LM (TK
The cells were inoculated and plaque cloning was performed 3 times to purify the clones. The vaccinia virus thus obtained is a recombinant vaccinia virus that has emerged by recombination with a plasmid vector DNA carrying NANBV cDNA, and has acquired the gene for β-galactosidase and is deficient in thymidine kinase. The results are shown in Table 8.

Table 8 Recombinant vaccinia virus and its properties Recombinant recombinant virus NANBV thymidine β-galacto vaccinia virus Genome kinase sidase clone plasmid (kb) activity used for production vUV17 pUV1 No No Yes vUV27 pUV1 No No Yes Yes ----------------------------------- VXE17 pxD39 Yes ) No Yes VXE28 pXE39 Yes (6.4) No Yes −−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−− vXX19 pXX49 Yes ( 9.0) No Yes vXX29 pXX51 Yes (9.0) No Yes vXX39 pXX51 Yes (9.0) No Yes

Step 3 (Detection and Confirmation of NANBV-Related Antigen Production by Recombinant Vaccinia Virus by Fluorescent Antibody Method) Detection and confirmation of recombinant vaccinia virus-produced antigen were performed by the indirect fluorescent antibody method. L cultured on cover glass
-M (TK) cells were inoculated with 10-fold diluted recombinant vaccinia virus and cultured by the method described in Reference Example 4. After 48 hours of culturing, take out the cover glass and set M / 7
After washing with 5 phosphate buffered saline pH 7.4 (M / 75 PBS) three times, it was washed once with distilled water and air-dried. Then −
Acetone was fixed at 20 ° C. for 5 minutes. The anti-NANBV mouse monoclonal antibody used as the primary antibody was used to immunize BALD / C mice by a conventional method with the core antigen, nonstructural protein antigens NS-3 and NS-5 prepared by Escherichia coli described in Reference Example 1. It was obtained from a hybridoma in which lymphocytes and mouse myeloma cells that were separated by fusion were fused.
A monoclonal antibody against the envelope was prepared by using, as an antigen, a 16mer consisting of the amino acid sequence of the amino acid sequence deduced from the envelope gene (1st to 16th positions on the N-terminal side) bound to bovine serum albumin.
In addition, the indirect fluorescent antibody method was performed by a conventional method. That is, primary cells were reacted with the acetone-fixed infected cells at 37 ° C. for 1 hour, washed with M / 75 PBS three times, and then fluorescent dye FITC-labeled anti-IgG (human or mouse) (Capel [Germany]
The product was reacted at 37 ° C. for 1 hour, washed 3 times with M / 75 PBS, and then examined under a fluorescence microscope. The results are shown in Table 9.

Table 9 Detection of NANBV-related antigen in recombinant vaccinia virus-infected cells by fluorescent antibody method Recombinant healthy human serum NANB hepatitis patient serum Vaccinia virus # 6 # 8 # 9 pool serum # II-1 pool serum # PS-1 Acute chronic vUV17 − − − − − − − vUV27 − − − − − − − vXE17 − − − + + + vXE28 − − − + + + + + vXX19 − − − + + + + vXX29 − − − + + + + + vXX39 − − − ＋ ＋ ＋ Monoclonal antibody Anti-core # 11 Anti-EnV # 755 Anti-NS3 # 74-1 Anti-NS5 # 8905 − − − − − − − − ＋ ＋ ＋ ＋ ＋ ＋ ＋ ＋ ＋ ＋ ＋ ＋ ＋ ＋ ＋ ＋ ＋ ＋ ＋ ＋ Seriously negative for normal human serum, non-A non-B hepatitis patient serum , A recombinant vaccinia virus clone vXE1 in which a part of the sequence of NS5 region of NANBV is deleted
7 and vXE28 also complete O protein of NANBV
RF clones vXX19, vXX29, vXX3
All 9 showed a positive reaction. When a mouse monoclonal antibody is used, anti-core antigen monoclonal antibody, anti-
For the envelope monoclonal antibody and the anti-NS3 monoclonal antibody, clones vXE17 and vXE2 were used.
8 and vXX19, vXX29, vXX39 all showed a positive reaction. For anti-NS5 monoclonal antibody, vXE1 in which the NS5 region of NANBV was deleted
7, vXE28 is negative, but vXX19, vXX2
9, vXX39 showed a positive reaction. These facts show that the target gene product is correctly expressed by the recombinant vaccinia virus, and particularly clone vXX19,
Infected with 29, 39, NANBV protein N
It is shown that the entire region covering from the core antigen on the terminal side to the NS5 protein on the C terminal side is expressed.

Step 4 (Analysis of recombinant vaccinia virus-infected cell culture supernatant by sucrose density gradient centrifugation) As described in Reference Example 4, recombinant vaccinia virus v
XX39 1.2 × 10 ⁷ PFU / ml of human liver-derived C
1.0 ml (1.2 x
(10 ⁷ PFU) was inoculated, the cells were adsorbed with the virus at 37 ° C. for 2 hours, and then the cells were cultured with M-199 alone at 37 ° C. for 3 days.
200 ml of the culture supernatant was centrifuged (3,000 xg, 5 ') and the supernatant was further centrifuged (48,000 xg, 14 hours, 4 hours).
℃), the precipitate was suspended in 2 ml of M / 75PBS, and sonicated (20 kHz, 200 W, 2 minutes).
Sucrose density gradient centrifugation from 0 w / v% to 60 w / v% (16
50,000 × g, 15 hours, 4 ° C.) for fractionation. Each fraction was sample buffer (final concentration: glycerol 20 v / v%, Tris.HCl 100 mM
pH 6.8, SDS 2w / v%, 2-mercaptoethanol 2v / v%, BPB 0.02w / v%), and after heating at 100 ° C for 5 minutes, 0.1w / v% SDS
-12.5% polyacrylamide gel electrophoresis. Next, the gel was subjected to Hybo analysis using a trans-blot apparatus (manufactured by Nippon Eido Co., Ltd.) to separate each protein separated by electrophoresis.
Blotting to an nd-ECL membrane (Amersham [UK]). Hybond-ECL membrane is 5 w / v
10 mM Tris.HCl p containing 10% skim milk
H7.2, 150 mM NaCl, 0.05 w / v%
Dip in a solution of Tween-20 (T-TBS) and incubate at room temperature for 1 hour to block. Then 50 with T-TBS buffer containing 1% skim milk.
A 0-fold diluted anti-core antigen monoclonal antibody (clone 29) was reacted at 37 ° C. for 1 hour, and 2 times well mixed with T-TBS containing 1% skim milk used for antibody dilution.
The ond-ECL membrane was washed and biotin-labeled anti-mouse I
gG (manufactured by Capel [Germany], diluted 500 times) is reacted at room temperature for 1 hour. The Hybond-ECL membrane was washed twice and the HRPO-labeled streptavidin (Amersha was used).
m company (UK), 500-fold dilution) is allowed to react at room temperature for 1 hour, the Hybond-ECL membrane is washed well with T-TBS four times, and an ECL western blotting detection system (Amersham [UK]) is used. And chemiluminescence reaction to Hyalbond-E on Saran wrap.
The CL film is wrapped, contacted with the X-ray film for 30 seconds, and then developed. As a result, the NANBV core antigen activity and the envelope antigen activity were 44 to 58 w / v in sucrose concentration.
% (FIG. 7), indicating that NANBV particles were obtained.

Step 5 (Electron Microscopic Observation of Recombinant Vaccinia Virus-Infected Cells) Recombinant vaccinia viruses vXX39 and vUV17 (the latter does not carry the NANBV genome) were transformed into human hepatocytes as described in Reference Example 4. Incubate for 2 days, collect the infected cells with rubber police, and use an epoxy resin (Nisshin EM
(Manufactured by K.K.), an ultrathin section was prepared, uranium / lead double staining was performed with 2% uranium acetate and lead citrate, and then observed with an electron microscope. As a result, N
Particles that were not observed in vUV17 infected cells without the ANBV genome were observed in cells infected with vXX39 (Figure 8). This result is combined with the result of step 4, and vX
It was shown that X39 produced NANBV particles.

[0095]

[Operation and effect of the invention]

(1) The NANBV particles of the present invention have an extremely wide antigenic spectrum as compared with conventional antigens, and not only chronic but also acute NA
Since it also exhibits an antigen-antibody reaction with the sera of patients with hepatitis NB excellently and specifically, it is possible to provide a highly reliable diagnostic agent having a high antibody detection rate and its accuracy. Further, the NAN of the present invention
When BV particles are used for screening blood for transfusion,
Blood infected with NANBV can be easily and reliably selected and removed from non-infected blood, so that blood-transmitted hepatitis NANB can be prevented. (2) The NANBV particles of the present invention can be advantageously used as an active ingredient of a highly immunogenic NANB hepatitis vaccine. In addition, a recombinant virus produced by inserting and linking NANBV genomic cDNA, for example, a recombinant vaccinia virus is useful as an active ingredient of a vaccine. (3) By using the NANBV particles of the present invention, N
Antibodies specific to ANBV, especially monoclonal antibodies,
This NANBV-specific antibody, which can be easily prepared, can be advantageously used not only as a diagnostic agent for detecting NANB hepatitis but also as an agent for removing NANBV from blood for transfusion. (4) Furthermore, it should be noted that the NANBV particles of the present invention are produced in an isolated state by gene expression of the DNA encoding the NANBV particles present in the host cell, not by viral infection of the animal. . Therefore, the possibility of infection at the production stage of NANBV particles is substantially eliminated, and the manufacturing cost can be reduced.
In addition, since all the materials used in the manufacturing process, for example, the medium used in the culture system are well known in terms of their composition, it is easy to purify and it is possible to obtain a highly pure NANBV particle product. (5) According to the present invention, it is possible to provide isolated NANBV particles of high concentration and high purity, which are not found in nature, and their genes. By using these NANBV particles and genes as pathogenic factors, non-A It will greatly contribute to the study of non-hepatitis B, hepatocellular carcinoma or liver cancer.

[Brief description of drawings]

1 and 1-2 show the NANBV gene cDNA used in the present invention for the entire region of the NANBV genome.
The relationship between A clones is shown.

FIGS. 2-1 to 2-16 are NAs used in the present invention.
The nucleotide sequence of the entire NBV gene cDNA region and the amino acid sequence encoded thereby are shown respectively.

FIG. 3 shows a comparison of the hydrophobicity index (hydrophobic / hydrophilic pattern) between the NANBV particles of the present invention and Japanese encephalitis virus (JEV). The horizontal axis represents the amino acid No. , The vertical axis is the hydrophobic index, the white triangle is the sugar chain binding site, and the asterisk is the common amino acid sequence of RNA polymerase (Gly-A
sp-Asp) site, C, M, E and NS are respectively
By core antigens, matrix antigens, envelope antigens, and nonstructural proteins.

FIG. 4 shows the NANBV genome c in animal cells.
The order of construction of the plasmid pMAM-neo10 for expression of DNA is shown.

FIG. 5: NANBV genomic cDNA in yeast
The order of construction of the plasmid pYHC5 for expression of A is shown.

FIG. 6 shows plasmids pXX-49, pXX-51 and pXX-39 for producing recombinant vaccinia virus.
The order of construction is shown respectively.

FIG. 7: Recombinant vaccinia virus vXX39
Of each fraction after sucrose density gradient centrifugation of the culture supernatant of
It shows sucrose concentration and antigenic activity respectively.

FIG. 8: Recombinant vaccinia virus vXX39
3 is an electron micrograph of NANBV particles produced in infected cells of E. coli.

[Procedure amendment]

[Submission date] September 25, 1991

[Procedure Amendment 2]

[Document name to be amended] Statement

[Correction target item name] 0007

[Correction method] Change

[Correction content]

(D) Diagnosis: As mentioned above, NANBV has not been identified yet, and no reliable viral marker for diagnosis is known. Therefore, finally, both hepatitis A and B,
Exclusion diagnosis based on the test of antibody titers of known respiratory viruses such as cytomegalo, EB, chickenpox, and herpes simplex, and histopathological diagnosis by liver biopsy are inevitable (S.
"Disease of the" by Shenlock
live and biliary system ”,
Eighth Edition, Pages 326-333, Blackwell
Scientific Publications 1
Published 989). In parallel with these, for example, serum enzyme, GPT [glutamate pyruvate transaminase; alias: ALT (alanine aminotransferase)], GOT [(glutamate oxaloacetate transaminase; alias: AST (aspartate aminotransferase)], guanine deaminase Measurement of increase in activities such as (alias: guanase) (hepatobiliary and pancreas, vol. 14,
Pp. 519-522, 1987); Diagnostic criteria based on the continuation of abnormally high GPT or GOT activity in serum as described above (Journal of Japanese Society of Transfusion, 31 (4), 316-).
320, 1985; Nippon Rinji, 46 , 2635-263.
8, 1988) and the like are adopted. Regarding immunological diagnosis, in the present situation where isolation and identification of NANBV is difficult as described above, it can be obtained by expressing a clone of the isolated NANBV cDNA by making full use of experience, technology and knowledge of genetic engineering and immunology. Antigen-antibody reaction between the antigens and NANB hepatitis patient serum has been adopted. For example, as an antigen, NA derived from plasma of a NANB hepatitis patient is used.
NBV cDNA expression product (European Patent Publication No. 3630)
25), an HCV cDNA expression product derived from hepatitis-induced chimpanzee plasma of NANB (European Patent Publication 318).
No. 216, or Tokuhyo 2-500880), NANBV
NANBV cDNA expression product derived from infected chimpanzee liver (European Patent Publication No. 293274, or Japanese Patent Laid-Open Publication No. Sho 6 (1999) -242242).
4-2576 and JP-A-1-124387) are known, and RIA (radioimmunoassay), EIA (enzyme immunoassay) and the like are commonly used for reaction systems. However, the antigenicities of such expression products have little commonality with each other, and, for example, the above-mentioned HCV C-10
In the case of 0 antigen, it is a tentative index and a guideline for chronic hepatitis due to HCV infection, but the reaction region exhibiting antigenicity is narrow, and the detection rate of antibody is about 70%, which is insufficient (microorganism, 5 , 463-475, 1989; hepatobiliary-pancreas, 20 , 4
7-51, 1990; History of Medicine, 151 , 871,
1989), broadly defined hepatitis NANB or NANBV
It is considered insufficient for definitive diagnosis of infection, progress and treatment of chronic hepatitis, and determination of acute hepatitis. Therefore, the diagnosis,
Further development of more detailed virological and immunological investigations is expected to ensure the determination of the course and treatment.

[Procedure 3]

[Document name to be amended] Statement

[Correction target item name] 0010

[Correction method] Change

[Correction content]

[0010]

The inventors of the present invention have conducted extensive studies to overcome the above-mentioned problems of the prior art, and as a result, 1
NANBV gene cDNA having a base number of 000 or more
The ORF region of the NANBV genomic cDNA from the C protein gene to the NS3 gene is linked to each other by linking 10 or less clones with their ORFs matched with each other, a region covering the longest NANBV total ORF longer than the region, and the entire ORF. Both non-cods at the 5'and 3'ends in the region
The NANBV genome whole region linked with the ing region was constructed accurately, and, surprisingly, the mass production and the acquisition of NANBV particles were achieved by inserting and expressing each such region in an expression vector. The NANBV particle referred to in the present invention means an expression product of the above-mentioned region of the NANBV genome, and contains at least one antigen selected from the group consisting of non-A non-B hepatitis virus core antigen, matrix antigen and envelope antigen. Including. Non-A
Examples of non-hepatitis B virus (NANBV) particles include the following structures: A NAN consisting mainly of C (core) antigen, M (matrix) antigen and E (envelope) antigen.
NABV complete particle having BV antigen assembly and nucleic acid inside the virus particle; NANBV antigen assembly mainly composed of C antigen, M antigen and E antigen, NANBV incomplete particle having no nucleic acid inside the virus particle; mainly A NANBV antigen assembly consisting of C antigen, and a NANBV core having a nucleic acid inside the core; a NANBV antigen assembly mainly consisting of C antigen,
NANBV incomplete core with no nucleic acid inside its core;
And a NANBV surface antigen assembly consisting mainly of E antigen. Such an achievement is directly (a
U.S.A.) for the purpose of extracting RNA, NANBV and its gene can be stored in a body fluid or blood without undergoing the amplification process of NANBV using a chimpanzee having an unknown transfer factor as a host and under the preservation of the gene isolation material. This is because the careful operation was carried out so as not to be cleaved / degraded by the enzyme therein and to obtain complete NANBV genomic RNA or a long-chain RNA fragment of about 2 kb to about 5 kb. In other words, the NANBV gene is cloned directly from fresh whole blood of a NANB hepatitis patient and excised liver strips of a liver cancer patient associated with NANB hepatitis, that is, a highly reliable human-derived fresh material containing the NANBV gene. Moreover, the expression was successful.
Furthermore, since the blood antigen amount of NANBV is remarkably low, which is about 10,000 times lower than that of both hepatitis A virus and hepatitis B virus, the problem that isolation and identification of the NANBV antigen is difficult was successfully solved. . That is, as described in detail later, after extracting and purifying RNA from the above-mentioned fresh material of human origin that has been strictly selected and collected, a cDNA library of such RNA is prepared using reverse transcriptase, and then the cDNA is prepared. Each cDNA from the library was cloned into a phage vector lambdagt
11 and each cDNA is expressed on a phage plaque at a high concentration, and the NANBV antigen is repeatedly detected by EIA using the expression product and acute NANB hepatitis patient convalescent serum and chronic NANB hepatitis patient serum. Have successfully screened a long-chain NANBV gene cDNA having about 1000 to about 5000 bases. Also, the NANBV genomic cDNA whole region and the NANBV genomic cDNA ORF whole region constructed by matching and linking the ORF whole regions of the cDNAs carefully selected from the cloned cDNA group are inserted into an expression vector and expressed. Because of this, N is
It was also found that the ANBV particle or NANBV antigen assembly can be obtained in large quantities and at low cost. In addition, the expression product of the present invention has a broader antigen spectrum than the expression products of conventional partial fragments of NANBV genomic cDNA, and is excellent in not only chronic but also acute NANBV hepatitis patient sera and specific antigen-antibody reaction. Therefore, it was found that the antibody detection rate is 95% or more, which solves all of the above problems (f). That is, it brings about a great progress in enhancing the immunogenicity of the vaccine, improving the detection rate of the antibody of the diagnostic agent, and producing the antibody. The present invention has been completed based on these findings. Therefore, it is an object of the present invention to provide isolated NANB hepatitis virus particles useful as an active ingredient of a diagnostic agent and vaccine for NANB hepatitis. The present invention also aims to provide a method for producing isolated NANBV particles. or,
The present invention aims to provide a diagnostic agent for NANB hepatitis. Moreover, this invention aims at provision of the vaccine of NANB hepatitis.

[Procedure amendment 4]

[Document name to be amended] Statement

[Correction target item name] 0014

[Correction method] Change

[Correction content]

In the present invention, unless otherwise specified, the left and right ends of the deoxyribonucleic acid sequence are the 5'end and the 3'end, respectively. Unless otherwise specified, the left and right ends of the amino acid sequence of a peptide are the N-terminus and C-terminus, respectively. Isolated NANBV of the present invention
Particles can be prepared and identified according to steps (I) to (VII) below. (I) Selection and collection of RNA extraction material of NANBV gene: NANBV RNA extraction material is NANB
V carrier or blood, lymph, ascites, hepatocytes, and NA of humans or chimpanzees affected with V
Hepatocellular carcinoma associated with hepatitis NB or hepatocytes of liver cancer patients can be used. Especially in the chimpanzee-derived material, N
The amount of ANBV is relatively small compared to that of humans, and NA
The use of human-derived material is desirable because it contains unknown factors that are considered difficult to separate NBV. blood,
Of the lymph, ascites, and hepatocytes, blood is most easily obtained in large quantities, for example, blood that cannot be used as blood for transfusion is available in large quantities from blood banks. Such blood can be advantageously used as a NANBV RNA extraction material. 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 the surface antigen of the hepatitis B virus (WHO expert committ).
ee on viral hepatitis: Adv
ances in viral hepatitis,
WHO Technical Report Seri
es, 602 , 28-33, 1977) and the gene DNA of hepatitis B virus (Brechot, C; Ha.
dchouel, M; Scotto, J; Degos,
F; Charnay, P; Trepo, C; Tioll
ais, P: Detection of hepati
tis B virus DNA in liver
and serum: a direct appraisal
sal of the chronic carrier
r state. Lancet, 2 , 765-768,
1981) and determine whether the plasma is a serum enzyme that has been adopted as a standard for the diagnosis of hepatitis NANB, such as GPT (Wrobleski,
F. & LaDue, J .; S. : Serum glut
amic-pyruvic transaminese
in cardiac and hepatic di
sease, Proc. Soc. Exp. Biol. M
ed. , 91 , 569, 1956. ), GOT, guanase, etc. The above-mentioned step of separating blood into plasma and red blood cells and inspecting the plasma is carried out for various bloods, and both the surface antigen of hepatitis B virus and gene cDNA are negative, and the activity of the enzyme is abnormally high. , For example, pool plasma with a GPT activity of 35 IU / l or more.

[Procedure Amendment 5]

[Document name to be amended] Statement

[Correction target item name] 0020

[Correction method] Change

[Correction content]

The cDNA library can be prepared by using commercially available kits, for example, cDNA cloning systems Lambda gt10 and Lambda gt11 [Amersham (UK), BRL (US), Stratagene (US)], in vitro packaging system. [Amersham (UK), BRL (US), S
manufactured by Tratagene (USA)] and the like.

[Procedure correction 6]

[Document name to be amended] Statement

[Correction target item name] 0021

[Correction method] Change

[Correction content]

(V) NAN from cDNA library
Cloning of cDNA containing BV gene: In this step, a cDNA clone containing NANBV gene is obtained. When the cDNA library contains transformants, these transformants are cultured in an agar medium to form colonies. On the other hand, in the case where the cDNA library contains recombinant phage particles or recombinant viruses, these recombinant phage particles or recombinant viruses may be used for known sensitive host cells such as Escherichia coli, Bacillus subtilis, yeast and animal cell cultures. After infection, the plaques are formed by culturing or infected cells are proliferated. And
Under known immunoassays, NANB regardless of acute NANB hepatitis convalescent serum, chronic NANB hepatitis patient serum, intrahepatic cytoplasmic tubular formation type or non-formation type
Using V.-infected chimpanzee sera, NANBV antigen-producing colonies, plaques or infected cells are selected and isolated by reacting with at least one of the above-mentioned sera. For the strict selection of colonies, the previous steps were repeated and from each isolated colony, plaque or infected cell, T. Molecu by Maniatis et al.
lar Cloning, A Laboratory
Manual (Cold Spring Labor)
story [U. S. A], 309-433, 198.
A cDNA clone containing the NANBV gene is isolated according to 2). The immunoassay can be performed by, for example, an enzyme-labeled antibody technique using an antibody labeled with the enzyme peroxidase or alkaline phosphatase, and a fluorescent antibody technique using an antibody labeled with fluorescein isothiocyanate or europium. In the immunoassay according to the above-mentioned technique, it is desirable to carry out the method by an indirect method capable of achieving a highly sensitive immunoassay even when a small amount of patient serum is used. In this indirect method, it is preferable to use serum of NANB hepatitis patients or NANB hepatitis chimpanzees, which have a relatively high content of antibodies specific to NANBV, as the primary antibody, and an enzyme or fluorescent substance as the secondary antibody. Labeled commercial anti-human Ig
(Immunoglobulin) antibody can be used.

[Procedure Amendment 7]

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[Name of item to be corrected] 0025

[Correction method] Change

[Correction content]

The 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
Confirm the position of AUG on A) and perform sequentially. For an important part of such an amino acid sequence, for example, a hydrophilic site that is considered to constitute an epitope, peptides corresponding to each hydrophilic site are synthesized, and high performance liquid chromatography (HP
After purification by LC), these peptides were analyzed by enzyme immunoassay (EIA) or radioimmunoassay (R).
It can be specified by subjecting it to IA).

[Procedure Amendment 8]

[Document name to be amended] Statement

[Name of item to be corrected] 0030

[Correction method] Change

[Correction content]

Regarding the homology, the nucleotide and amino acid sequences of the NANBV gene and various known viral genes (Japanese Patent Laid-Open No. 62-286930; Virolog) are used.
y, vol. 161, 497-510, 1987.
Year), eg bovine viral diarrhea virus (Viro
LOGY 165, 497-510, 198.
8 years), Swine fever virus (Virology, 17th)
1, 555-567, 1989), Tobacco Leaf Mottle Virus (Nucleic Acid Reseal)
arch, volume 165, pages 5417-5430, 1
986) and other viral genes.

[Procedure Amendment 9]

[Document name to be amended] Statement

[Name of item to be corrected] 0032

[Correction method] Change

[Correction content]

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 indexes of both viruses are compared with each other. In addition, various antigen proteins of NANB virus particles,
That is, each gene region encoding viral core antigen protein (C antigen), matrix antigen protein (M antigen), envelope antigen protein (E antigen), and 6 types of nonstructural proteins (NS proteins) is encoded by it. Hydrophobicity indices between each peptide and known flaviviruses are compared, and the amino acid sequences of such peptides are compared with signal peptidases derived from host cells (Journal of Molecular).
r Biology, 167 , 391-409, 198.
3) and a known serine protease derived from flavivirus (Virology, 171 , 637-639, 19).
89) can be determined by comparing with the peptide binding site on which 89) acts. The NANBV genome of the present invention can be shown as follows using the base numbers disclosed in FIGS. 2-1 to 2-16: C protein is the third
33 to 677, M protein is 678 to 90
No. 5, E protein No. 906 to No. 1499, NS1 No. 1500 to No. 2519, NS2 No. 2520 to No. 3350, NS3 No. 3351 to No. 517
7th, NS4a is 5178th to 5918th, NS
4b is number 5919 to 6371, and NS5 is number 6372 to 9362. It can be pointed out that each antigen protein encoded by each region of these genes is useful as an antigen for a NANB hepatitis vaccine or diagnostic agent. In addition, since the NANBV particles of the present invention have a wide variety of epitopes, a diagnostic agent using such NANBV particles or NANBV antigen assembly as an antigen is more susceptible to NANB hepatitis than a diagnostic agent having a specific epitope region as an antigen. The specific reaction range (reaction spectrum or antigen spectrum) with a wide variety of anti-NANBV antibodies induced under morbidity is wide, and the sensitivity and accuracy of antibody detection as a diagnostic agent are extremely high, as illustrated in Examples below. It turns out to be expensive.

[Procedure Amendment 10]

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[Correction method] Change

[Correction content]

(VI) Expression of the entire NANBV genomic cDNA region and ORF region, and NANBV antigen assembly, NANBV incomplete particles and infectious NANB
Large-scale production of V complete particles: This step is carried out to express the NANBV gene cDNA cloned in (V) above in large amounts for industrial use. First, the entire region of each cDNA clone of the NANBV gene or a portion thereof is cut out with a restriction enzyme to obtain the NANBV gene cDNA.
After preparing the A fragment, the cDNA fragment is linked and then inserted into an expression vector. That is, NANBV genome cDNA
To construct the entire region of A and the entire ORF, clone c
In order to save labor in linking DNA fragments and to avoid linking errors, 10 or less or 5 or less cDNAs
The use of fragments is preferred. For that purpose, a long-chain cDNA having a DNA base number of 1000 or more, preferably 1500 or more
A clones are carefully selected, and the ORFs of these cDNAs are matched with each other and linked. For example, the NA disclosed in FIGS.
Among NB cDNA clones, BK112 and BK14
By using 6, BK147, BK157 and BK166, the above entire region can be constructed. In addition, known or commercially available expression vectors, for example, the plasmid vector pSN508 of the family Enterobacteriaceae (US Pat. No. 4,
703, 005), yeast plasmid vector pBH.
103 series (JP-A-63-22098) and pJM105
(JP-A-62-286930), vaccinia virus W
R strain (ATCC VR-119), LC16m8 strain (Japanese Patent Publication No. 55-23252), attenuated chickenpox virus Oka strain (US Pat. No. 3,985,615), attenuated Marek's disease virus (Journal of Japanese Veterinary Medicine, Vol. 27, 20-24 pages, 1
974; and Gan Monograph on
Cancer Research, Volume 10, 91-1
07, 1971), pTTQ series [Amers
manufactured by ham (UK)], pSLV [Pharmacia
LKB (Sweden)] and the like can be used. Then, an expression vector in which the NANBV gene cDNA is linked and linked is transferred or transfected into a host cell sensitive to the vector by a conventional method, and a transformant or infected cell producing NANBV particles can be selected and collected.
The presence or absence of NANBV antigen production can be detected in the same manner as in the immunoassay described in (V) above. Also, NA
The NBV particles can be confirmed or measured by a conventional method such as electron microscopy, immunoelectron microscopy, density gradient centrifugation or light scattering. As a result, when a plasmid was used as an expression vector, a transformant producing NANBV particles was obtained, and when an animal viral gene, that is, a viral vector was used, it was found, for example, in European Patent Publication No. 334530. Such a recombinant virus can be obtained.
Then, such transformants and recombinant viruses are respectively cultured by a conventional method, and homogeneous NANBV is contained in these cultures.
The particles can be produced in large quantities.

[Procedure Amendment 11]

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[Correction target item name] 0034

[Correction method] Change

[Correction content]

(VII) Purification of NANBV particles: Purification of NANBV particles in the culture obtained in the above (I) can be carried out by a known method such as salting out, adsorption / desorption method using silica gel or activated carbon, organic solvent. Precipitation method, fractionation method by ultracentrifugation, separation method by ion exchange and affinity column chromatography, high performance liquid chromatography,
This can be carried out by appropriately combining and adopting a fractionation method by electrophoresis and the like. When NANBV particles are purified and collected from a culture of a transformant such as Escherichia coli or yeast, contamination with allergens derived from E. coli or yeast will reduce the quality of the final product. Treatment, adsorption and desorption of impurities by activated carbon, and fractionation by density gradient centrifugation are performed sequentially (JP-A-63-29).
7) is desirable. In the case of purifying and collecting NANBV particles from a recombinant virus culture, for example, a cell culture medium, highly pure NANBV particles can be collected by repeating ultracentrifugation or density gradient centrifugation. You can Furthermore, in order to detoxify NANBV particles in the culture to ensure safety and to fix and stabilize the immunogenicity or antigenicity of the antigen, the culture before purification or after sterilization thereof A conventional inactivating agent such as formalin is added to the culture solution at a final concentration of 0.0001 to 0.001.
% (V / V), and then mixed at 4-37 ° C for 5
Inactivation of NANBV antigen by storage for 90 days is desirable. However, the recombinant attenuated virus can be used as an active ingredient of a live vaccine without inactivating it. Such a highly purified NANBV particle suspension can be used as a NANBV particle stock solution (NANBV vaccine stock solution) for the production of vaccines and diagnostic agents.

[Procedure Amendment 12]

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(VIII) Key Points and Main Uses of the Present Invention: According to yet another aspect of the present invention, (a) 1000 prepared from a non-A non-B hepatitis virus genomic RNA fragment of at least 1000 bases. 1 with each of the above base numbers
0 or less different cDNA clone groups are provided, wherein each clone cDNA of the cDNA clone group is provided.
The group as a whole covers at least the region of 333 to 5177 of the non-A non-B hepatitis virus whole nucleotide sequence of nucleotide sequences 1 to 9416 shown in FIGS. 2-1 to 2-16. And (b) a cDNA fragment group each having a base sequence determined in advance so that the base sequence obtained when sequentially arranged has at least a region corresponding to the region of base numbers 333 to 5177. The cDNA clone was excised from the cDNA clone, and (c) the c having each of these predetermined nucleotide sequences
2-1 to FIG. 2-16, which are obtained by sequentially connecting DNA fragment groups.
Constructing a first deoxyribonucleic acid containing at least the nucleotide sequence of nucleotide numbers 333 to 5177 of the non-A non-B hepatitis virus whole nucleotide sequence of nucleotide numbers 1 to 9416 shown in (d) A first deoxyribonucleic acid and at least one deoxyribonucleic acid selected from the second deoxyliponucleic acid obtained by substituting at least one base of the base sequence of the first deoxyribonucleic acid by degeneracy of the genetic code, a plasmid and an animal. When the replicable expression vector is a plasmid, a replicable recombinant DNA having the plasmid and at least one deoxyribonucleic acid introduced thereinto is introduced into a replicable expression vector selected from viral genes. Or, if the expression vector is an animal virus gene, the animal virus gene and its introduction A recombinant virus having at least one of the deoxyribonucleic acid selected from the above, and (e) when the replicable expression vector used in step (d) is a plasmid, the recombinant DNA is transduced into a prokaryotic cell or a eukaryotic cell. To form a transformant, and then the transformant is sorted and collected from a prokaryotic or eukaryotic cell culture, and the transformant obtained in (f) step (e) is a prokaryotic or eukaryotic cell. To produce non-A non-B hepatitis virus particles by expressing the deoxyribonucleic acid in the medium, or culturing the recombinant virus obtained in step (d) to cultivate the animal virus gene and the deoxyribonucleic acid. Recombinant virus containing non-A non-B hepatitis virus particles expressed by expressing animal virus and non-A non-B hepatitis virus particles expressed therewith Produced in the form,
And (g) there is provided a method for producing non-A non-B hepatitis virus particles, characterized in that the non-A non-B hepatitis virus particles are isolated alone or in the form of the amplified recombinant virus.

[Procedure Amendment 13]

[Document name to be amended] Statement

[Correction target item name] 0040

[Correction method] Change

[Correction content]

The purified NANBV particles of the present invention are N
It is useful as a diagnostic agent for hepatitis ANB. Further, the NANBV particles of the present invention can be prepared as a diagnostic agent as follows. Purified NAN obtained in the above (VII)
The BV particle solution is dispensed into a vial or a container such as an ampoule and then sealed. The injected particles can be lyophilized by the same means as described above before sealing, and the amount of NANBV particles in the container is generally about 1 μg to about 10 mg. Alternatively, the NANBV particles can also be adsorbed to the surface of commonly used supports such as microplates, polystyrene beads, filter papers or membranes.

[Procedure Amendment 14]

[Document name to be amended] Statement

[Correction target item name] 0041

[Correction method] Change

[Correction content]

The determination of the reactivity between serum and the NANBV particles can be individually carried out by the same means as described in (V) above. That is, the determination of such reactivity is determined by conventional immunoassay methods, such as radioimmun.
oassay (RIA), enzyme-linked
immunosorbent assay (ELIS
A), fluorescent antibody method (FA), passive red blood cell collection reaction (PH
A), reverse passive red blood cell collecting reaction (rPHA), etc. can be used. The amount of the NANBV particles used in the above immunoassay is about 0.1 to about 100 mg / m 2.
The amount used for RIA, ELISA, FA, PHA and rPHA is 0.1 to 1 mg / m 2, respectively.
1, 0.1-1 mg / ml, 1-100 mg / ml, 1
~ 50 mg / ml and 1-50 mg / ml.

[Procedure Amendment 15]

[Document name to be amended] Statement

[Name of item to be corrected] 0045

[Correction method] Change

[Correction content]

The NANBV vaccine stock solution prepared in the above (VII) was filtered by a conventional method using a microfilter to sterilize the solution, and then the filtrate was Low with a physiological salt solution.
The protein concentration measured by the ry method is about 1 to about 500 μg /
Dilute to a final volume of ml and use aluminum hydroxide gel as an adjuvant to give a final concentration of about 0.1 to about 1 mg / ml.
To be added. Furthermore, at least one stabilizer can be added to such a mixture, and a commercially available known stabilizer can also be used as the stabilizer, for example, gelatin and its hydrolyzate, albumin, saccharide glucose, Fructose, galactose, sucrose and lactose, as well as the amino acids glycine, alanine, lysine, arginine, glutamine and the like can be used. As the adjuvant, calcium phosphate gel, aluminum phosphate gel, aluminum sulfate, alumina, and bentonite which are precipitation adjuvants, and a muramyl peptide derivative which is an adjuvant for inducing antigen production, polynucleotide, Krestin [registered trademark, Kureha Chemical Industry Co., Ltd. (Japan ) Production and sales] and Picibanil (both are antitumor agents) can also be used.

[Procedure Amendment 16]

[Document name to be amended] Statement

[Correction target item name] 0046

[Correction method] Change

[Correction content]

Then, the NANB hepatitis vaccine solution containing the gel-adsorbed NANBV particles is dispensed into a small container such as an ampoule or a vial and sealed to obtain NANBV.
A purified precipitated NANB hepatitis vaccine containing particles is obtained.

[Procedure Amendment 17]

[Document name to be amended] Statement

[Correction target item name] 0047

[Correction method] Change

[Correction content]

By freeze-drying the thus 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, in the tropics. Such a dried preparation is generally prepared by dispensing a liquid precipitated NANB hepatitis vaccine into a container such as a vial and an ampoule, followed by a conventional method. After freeze-drying, nitrogen gas is injected into the container containing the dried vaccine and the container is sealed. Regarding the quality of vaccine preparations, "Precipitation B" specified in the Japanese Ministry of Health and Welfare Notification No. 159 "Standards for Biological Products"
Test according to "Hepatitis C Vaccine", "Dried Japanese Encephalitis Vaccine" and "Precipitated Pertussis Vaccine".

[Procedure 18]

[Document name to be amended] Statement

[Correction target item name] 0049

[Correction method] Change

[Correction content]

Generally, the vaccine containing NANBV particles of the present invention is injected into a vial such as an ampoule and sealed, and
It can be provided in liquid, suspension or dried form.
When a dried vaccine is used, it is dissolved or suspended in sterile distilled water so as to have the original volume before freeze-drying before use. Generally, this vaccine is used by subcutaneous inoculation, and the amount of vaccination for an adult is about 0.5 ml, but a child is vaccinated with half the dose of an adult. In addition, such a vaccine is usually administered twice at intervals of about 1 week to 1 month,
Approximately half a year later, one additional vaccination will be given. In the case of a live vaccine, it is usually sufficient to give one dose.

[Procedure Amendment 19]

[Document name to be amended] Statement

[Correction target item name] 0050

[Correction method] Change

[Correction content]

Further, the NANBV particles can be used for producing antibodies such as polyclonal antibodies and monoclonal antibodies specific to the NANBV particles. For example, the NAN
A polyclonal antibody specific for BV particles can be prepared by the following conventional method. First, the purified NANBV particles of the present invention are added subcutaneously, intramuscularly, intraperitoneally or intravenously to animals such as mice, guinea pigs and rabbits, usually 1 to 4 times.
Inoculate several times at weekly intervals to fully immunize such animals. Conventional, commercially available adjuvants can be used to enhance the immune effect. Then, serum was collected from the immunized animal, and then anti-NANBV was collected from the serum according to a conventional method.
The particle polyclonal antibody is isolated and purified.

[Procedure amendment 20]

[Document name to be amended] Statement

[Correction target item name] 0051

[Correction method] Change

[Correction content]

On the other hand, the monoclonal antibody specific to the NANBV particles is, for example, described in "Cell Engineering" Vol.
It can be prepared by a conventional method described on page 9 (1982). For example, splenocytes obtained from mice immunized with purified NANBV particles and commercially available mouse myeloma cells were fused by a cell fusion technique to prepare hybridomas, and then the NANs were prepared from these hybridomas.
A hybridoma is obtained that has the ability to produce antibodies that react with BV particles. This is cultured by a conventional method, and the anti-NANBV particle monoclonal antibody is isolated and purified from the culture supernatant.

[Procedure correction 21]

[Document name to be amended] Statement

[Correction target item name] 0052

[Correction method] Change

[Correction content]

The above-mentioned polyclonal antibody and monoclonal antibody can also be used as a diagnostic agent for NANB hepatitis. NANB using such antibodies
Diagnosis of hepatitis B can be carried out in substantially the same manner as the above-mentioned method for diagnosis of hepatitis NANB using the NANBV particles. By using a polyclonal antibody or a monoclonal antibody, it is possible to identify and quantify the NANBV particles present in liver tissue and blood. Further, the polyclonal antibody and the monoclonal pile specific to the NANBV particles of the present invention can be used as a NANBV removing agent for blood for transfusion. That is,
NANBV present in blood can be effectively removed, for example, by affinity chromatography techniques using polyclonal or monoclonal antibodies.

[Procedure correction 22]

[Document name to be amended] Statement

[Correction target item name] 0054

[Correction method] Change

[Correction content]

Example 1 (Part I) Step 1 [DNA complementary to NANBV genomic RNA
Preparation of Plasma-Derived RNA to Produce (cDNA)] To obtain NANBV from plasma, blood GPT value (Wrob
lewski, F & J. S. LaDue: Serum
glutamic-pyruvic transam
inase in cardiac and hepa
tic disease. Proc. Soc. Exp.
Biol. Med. , 91: 569, 1956) is 3
Sucrose 30% with 4.8 L human plasma showing 5 IU / L or more
(W / W) After layering on the solution, centrifugation (48,000 xg, 1
3 hours, 4 ° C.), and the precipitate was washed with 50 mM Tris. HC
1, pH 8.0-1 suspended in EDTA solution,
After centrifugation (250,000 × g, 3 hours, 4 ° C.) again, the resulting precipitate was added to 75 ml of 5.5M GTC solution (5.5
M guanidine thiocyanate, 20 mM sodium citrate, pH 7.0, 0.05% sarcosyl, 0.1
M2-mercaptoethanol) and dissolved in 16 ml of CsTFA-0.1M EDTA solution (ρ = 1.51).
Layered on top. Centrifugation (140,000 xg, 20 hours,
15 ° C) to precipitate RNA. Protein and DNA in the supernatant are removed by aspiration, and the precipitate is removed with 200 μl TE (1
0 mM Tris.HCl, pH 8.0, 1 mM ED
TA) solution, add 20 μl of 3M sodium chloride and ethanol, and leave at −70 ° C. for 90 minutes. Centrifuge (12,000 xg, 30 minutes, 4 ° C), and precipitate with TE.
, Sodium chloride and ethanol were added again in the same manner as above, the mixture was allowed to stand at -70 ° C, and the resulting precipitate was recovered,
It was dissolved in 10 μl of TE to obtain purified RNA.

[Procedure amendment 23]

[Document name to be amended] Statement

[Correction target item name] 0067

[Correction method] Change

[Correction content]

Next, the phages of the cDNA library obtained in step 5 were L-ligated as described in step 7.
After incubation in agar medium at 42 ° C. for 3 hours and then at 37 ° C. for 3 hours, the mixture is cooled, put on a nitrocellulose filter and left for 30 to 60 seconds to adsorb the phage to the filter. Alkali denaturation (0.5N sodium hydroxide-1.5M sodium chloride solution for 1-5 minutes),
Neutralization (0.5 M Tris.HCl pH 8.0-1.
After treatment with 5M sodium chloride solution for 1 to 5 minutes), 2 x SSC
It is washed with (0.3 M sodium chloride-0.03 M sodium citrate solution), air-dried, and baked at 80 ° C. for 2 hours. Then filter the hybridization solution (5
0% formamide, 5 × SSC, 5 × Denhart
Solution, 50 mM phosphate citrate buffer pH 6.5, 1
4 with 00 μg / ml salmon sperm DNA, 0.1% SDS)
After incubating at 2 ° C. for 6 hours, about 4 × above
Immerse again in 300 ml of the above hybridization solution containing 1 ml of 10 ⁸ cpm / ml probe at 42 ° C.
Incubate for 16-20 hours and wash (2 x SS
C-0.1% SDS solution 4 times, 0.1 × SSC-0.1
% SDS solution twice), followed by drying and autoradiography to isolate clones showing positive hybridization. As a result, 27 clones that react with the probe derived from BK102, 14 clones that react with the probe derived from BK106, and 13 clones that react with the probe derived from BK112 could be obtained. Each clone was sequentially named as BK114 to BK169. Furthermore, when the nucleotide sequence was determined as described in step 8 and mapping of each clone was performed, it was mapped to a size of about 9.5 Kb, which is considered to be almost the entire length of the NANBV genome (Fig. 1-2). Of this, a 0.55 Kb fragment on the 5'side from clone BK157 located on the 5'end side was recovered after digestion with the restriction enzyme KpnI, and was also found on the 3'side of clone BK116 located on the 3'most end side and 0.55 Kb. Fragment was recovered after digestion with restriction enzymes Hpal and EcoRI, a probe labeled with ³² P as described above was prepared, and phages of the cDNA library obtained in step 5 were plaque-hybridized and newly prepared with a probe derived from BK157. 3 clones were isolated in
It was named K170, BK171 and BK172.

[Procedure correction 24]

[Document name to be amended] Statement

[Correction target item name] 0069

[Correction method] Change

[Correction content]

Reference Example 1 [Production of Various Antigens Associated with Antibody Responses Associated with NANBV Infection (hereinafter referred to as NANBV-Related Antigens) by E. coli] BK106, BK in Step 8 of Example 1 (Part I)
The plasmid DNA in which the cDNA of each of 111, BK112 and BK147 obtained in step 9 of Example 1 (Part I) was inserted was recovered by the alkaline method. Then, the DNA of BK106 was digested with restriction enzymes EcoRI and Cla.
Of the 0.34 Kb DNA fragment digested with I and recovered.
5 μg of T4 DNA polymerase reaction solution (67 mM T
ris HCl pH 8.8, 6.7 mM magnesium chloride, 16.6 mM ammonium sulfate, 10 mM 2-mercaptoethanol, 6.7 μM EDTA, 0.02%
Bovine serum albumin, 0.3 mM dNTP and 2-5
Unit T4 DNA polymerase) at 37 ° C. at 60
Minutes, and both ends were blunted. BK10
The DNA of 2 was digested with the restriction enzyme BamHI, and 0.5 μg of the recovered 0.7 Kb DNA fragment was treated with T in the same manner as above.
The ends were made blunt using 4 DNA polymerase. BK
The 147 DNA was digested with the restriction enzyme Sau3AI, and 0.5 μg of the recovered 1 Kb DNA fragment was blunt-ended in the same manner as above. The BK111 DNA was digested with the restriction enzyme EcoRI, and 0.5 μg of the recovered 1 Kb DNA fragment was blunt-ended in the same manner as above. Next, the expression vector pKK233-2 (Amann, E. an.
d J. Brosius. ATG vector fo
r regulated high-level ex
compression of cloned genes
in Escherichia coli. Gene,
40 , 183, 1985) to the restriction enzyme Hind
After digestion with III, 2 μg of DNA was incubated in S1 nuclease reaction solution (0.3 M sodium chloride, 50 mM sodium acetate pH 4.5, 1 mM zinc sulfate, 100 to 200 units of S1 nuclease) at 37 ° C. for 20 minutes. , 0.12M EDTA and 1M Tris HC
The reaction was stopped by adding 1/10 volume of pH 9.0 solution, phenol extraction was performed, and vector DNA having blunt ends was precipitated with ethanol and recovered. The DNA of the vector pKK233-2 was digested with the restriction enzyme PstI, purified by phenol extraction and alcohol precipitation, and then 2 μg was obtained by the above T4 DNA polymerase reaction.
The ends cleaved by PstI of the vector DNA of 1. were blunted. The clones BK106 and BK thus obtained
The 111-derived DNA fragment was blunt-ended after vector cleavage with HindIII, and the clones BK102 and BK147-derived DNA fragments were Ps and Ps, respectively.
After cleavage with tI, mix with vector DNA with blunt ends (0.5 μg of insert cDNA fragment and vector DNA
Each) 2 μl of 10 × ligation solution (500 mM
Tris.HCl pH 7.5, 100 mM magnesium chloride, 100 mM DTT, 10 mM ATP),
300 to 400 units of T4 DNA ligase and distilled water were added to make a total volume of 20 μl, and the mixture was incubated at 14 ° C. for 12 to 18 hours.
It was named E-06, pE-11, pB-02, pS-09. Next, E. coli JM109 strain was transformed with the above plasmid DNA as described in step 6 of Example 1 (Part I) to obtain transformed E. coli. Escherichia coli obtained in this manner was LB medium [1% (W / V) tryptone,
0.5% (W / V) yeast extract, 1% (W
/ V) sodium chloride, pH 7.5] at 37 ° C., IPTG (1 mM) is added in the logarithmic growth phase, and the mixture is further cultured for 3 hours. Centrifuge the cells (10,000 xg,
For 15 minutes), 50 mM Tris.HCl pH8.
0, sonicate (20 KHz, 600 W, 5 minutes), and then centrifuge (10,000 xg, 15 minutes) to fractionate into supernatant and precipitate, each containing sample buffer [2
0% (V / V) glycerol, 0.1M Tris.H
Cl pH 6.8, 2% (W / V) SDS, 2% (V / V
V) 2-Mercaptoethanol, 0.02% BPB]
Dissolved in water and heated at 100 ° C for 3 minutes, then 0.1% SDS
-After electrophoresis on 7.5% polyacrylamide gel, the protein was trans-blotted [BIO-RAD (USA)]
Manufactured] to a nitrocellulose filter. Then, the filter is immersed in 3% gelatin solution, left for 60 minutes, incubated with 100 times diluted serum of NANB hepatitis patient (room temperature, 2 to 3 hours), washed with water, and then TTBS solution (0.02M Tris.HCl pH7). .5, 0.5
M sodium chloride, 0.05% (V / V) Tween2
Wash with 0). Next, it is immersed in a 2000-fold diluted peroxidase-labeled anti-human IgG antibody solution and incubated at room temperature for 90 minutes. After washing with distilled water, wash with TTBS. Next, as described in Step 7 of Example 1 (Part I), the color former DAB and a 30% hydrogen peroxide solution of the substrate are immersed in the buffer added for 5 to 30 minutes and washed with water to stop the reaction.

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[0073]

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Recombinant E. coli JM109 (DE3) / pJ
As described in Reference Example 1, F-22 was cultivated in LB roasted medium, added with 0.5 mM IPTG, and further cultivated for 3 hours, and then collected to collect 2% SDS-2% 2-. After heating in a mercaptoethanol-containing buffer at 100 ° C for 3 minutes,
Electrophoresis was performed on a gel composed of 0.1% (w / v) SDS-1.25% (w / v) polyacrylamide, and the proteins separated on the gel were subjected to nitro by a trans-blot apparatus (manufactured by Japan Eido Co.). After blotting on a cellulose membrane, the protein produced by Western blotting was analyzed. The specific antiserum used in the western blot was obtained by purifying the NANBV-related antigen prepared in Reference Example 1 and immunizing a guinea pig. As a result of this analysis, it was shown that the protein produced by recombinant JM109 (DE3) / pJF-22 reacts with all antisera (No. 5).
table). Recombinants are derived from the core antigen encoded at the 5'end of the genome to the nonstructural protein NS5 encoded at the 3'end.
It was shown to express all the genes up to. Such a recombinant not only provides a very useful raw material for a diagnostic agent for NANBV infection, but also a raw material for a vaccine.

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Step 3 (NANBV whole gene cDNA
Production of NANBV Particles by Expression in a Animal Cell) The plasmid pORF-24 obtained in Step 1 was transformed with XbaI.
After digestion with, this was subjected to low melting point agarose gel electrophoresis to recover a DNA fragment of about 9 kb. Next, NheI
Plasmid cleaved with pMAM-neo (Clonte
The expression plasmid pMAM-neo10 was constructed by inserting and ligating into ch company (purchased from USA) (FIG. 4). The plasmid is then transferred to the calcium phosphate method ("Molecular Cloning", 16.
33-16.39, Cold Spring Harb
or Laboratory, 1989), human liver-derived cells, Chang Liver (ATCC).
CCL13) and chimpanzee liver-derived cells (purchased from Dainippon Pharmaceutical Co., Ltd.).
The cells into which the plasmid pMAM-neo10 was introduced were transformed into the antibiotic aminoglycoside G418 resistance, and G4
Since colonies were formed in the presence of 18 600 μg / ml, transformants were selected and cloned using this as an index. Transformed cell clones HL-A1 and HL-A2 obtained from human-derived cells, and transformed cell clone CL-B1 obtained from chimpanzee liver-derived cells
1. Cover glass placed in a petri dish at 37 ° C. for 4 days in Eagle MEM medium containing 5% (v / v) fetal bovine serum in the same manner as described in Reference Example 4 below with CL-B14. It was cultured on. The protein produced by cell culture was assayed for the presence or absence of various NANBV antigens by the conventional indirect fluorescent antibody method using the specific antiserum described in Step 2. As a result, the protein produced in the G418-resistant cell clone reacted with all antisera of guinea pigs immunized with the NANBV-related antigen prepared in Reference Example 1 (Table 6).

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Step 4 (Human or chimpanzee hepatocyte-derived transformed cell clones HL-A1 and CL-B
Sucrose density gradient centrifugation of NANBV-related antigen produced by 11) Clones HL-A1 and CL-B11 were placed in a carbon dioxide incubator at 37 ° C., 4 ° C. in 5 petri dishes for tissue culture each having a diameter of 9 cm, as described in step 3. Cultured for a day. Remove cells with a rubber policeman, pool with culture medium, sonicate (20 kHz, 200 W, 2 minutes), and centrifuge (500
The supernatant obtained by 0xg, 15 minutes, 4 ° C) was further centrifuged (48.0).
(00 × g, 14 hours, 4 ° C.), and the precipitate was mixed with 1 ml of M / 7.
Float in 5 PBS and sonicate for 2 minutes
Fractionation was performed after performing 0% (w / v) to 60% (w / v) sucrose density gradient centrifugation (160,000 × g, 15 hours, 4 ° C.). Each fraction was subjected to SDS by the method described in step 2.
-After polyacrylamide electrophoresis, core and envelope antigens were detected by Western blotting. As a result, both antigens had a sucrose density of 40% (w / v) to 50%.
It was detected in the vicinity of (w / v), indicating that NANBV particles were obtained.

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Example 2 Step 1 (Construction of plasmid for expression of NANBV whole gene cDNA by yeast and production of recombinant yeast) The plasmid pORF24 obtained in Example 1 was digested with XbaI to prepare the plasmid of Example 1 (Part II). Similar to step 1, a NANBV cDNA fragment of about 9 kb was recovered. This cd
0.5 μg of NA fragment was added to T4 DNA polymerase reaction solution (67 mM Tris.HCl pH 8.8, 6.7 m).
M magnesium chloride, 16.6 mM ammonium sulfate,
10 mM 2-ME, 6.7 mM EDTA-2Na
0.02 %% (w / v) bovine albumin, 0.3 mM
2 to 5 units of T4 DNA polymerase (manufactured by Takara Shuzo Co., Ltd.) was added, and the mixture was incubated at 37 ° C. for 60 minutes to make both ends blunt. Next, the XhoI linker (CCTCGAGG) is ligated with T4 DNA ligase. That is, 0.3 μg of DNA was added to 21 μl of 1
0x ligation solution (500 mM Tris-HCl
pH 7.5, 100 mM magnesium chloride, 100 m
MDTT, 10 mM ATP), 300-4
00 units of T4 DNA ligase (Takara Shuzo) and distilled water were added to a total volume of 210 μl, and the mixture was incubated at 14 ° C. for 12 to 18 hours. CDNA prepared in this way
The fragment was inserted and ligated into the XhoI site of the yeast expression vector YEp133PCT (US Pat. No. 4,810,492) to obtain the expression plasmid pYHC5 (FIG. 5). With this expression plasmid pYHC5, the yeast S. cerevisiae (ATCC No. 4477
2) by the alkali cation method (Ito, H. et al.
J. Bacteriol. , 153: 163-1681
983) to obtain recombinant yeast YHC5-1. This recombinant plant is designed to produce NANBV-related antigens by activating the yeast inhibitory acid phosphatase gene promoter when the phosphate ion in the medium is deficient and causing transcription of NANBV cDNA knotted downstream. ,
Has been built.

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[0085] Further, when sucrose density gradient centrifugation was performed in the same manner as in step 4 of Example 1 (Part II), both the core antigen and the envelope antigen were 40% (w / v) to 50% (w).
/ V) was detected in the sucrose concentration fraction, indicating that NANBV particles were obtained.

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Reference Example 4 (Culture of vaccinia virus WR strain) Vaccinia virus was cultured according to a conventional method. That is, mouse-derived thymidine kinase (TK) -deficient cell line LM cultured in a monolayer
(TK) (Original ATCC strain number CCL-1.3, Dainippon Pharmaceutical Co., Ltd.), monkey kidney-derived Vero cells or adult liver-derived cells Chang River (original ATCC strain number C)
CL-13, Dainippon Pharmaceutical Co., Ltd., etc.) were inoculated with 0.5 ml of vaccinia virus into cells cultured in a 6 cm dish and left at 37 ° C. for 1 to 2 hours to remove the virus solution. Then, 5 ml of Eagle's MEM medium (Nissui Pharmaceutical Co., Ltd.) supplemented with 5% (v / v) fetal bovine serum was added to the virus culture solution, and the cells were cultured at 37 ° C. for 24 to 48 hours, and when sufficient cell degeneration occurred. , Virus culture solution or infected cells were collected and suspended in MEM, and then sonicated (20 kHz, 200
W for 2 minutes) to obtain a virus solution.

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Reference Example 5 (Infectious titer measurement of vaccinia virus by plaque method)
The plaque method was performed according to a conventional method. That is, 0.1 ml / 6 of the virus solution of Reference Example 4 diluted 10-fold with M-199 (Sigma [USA] Inc.) was added to the cell culture described in Reference Example 4.
cm Petri dish, incubate at 37 ° C. for 2 hours, adsorb virus to cells, remove inoculum and add agar-containing medium (M-199 to fetal calf serum 3% (v / v), NaHC
O ₃ 0.14% (w / v) and agar 0.8% (w / v) were added) at 5 ml / si.

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Step 4 (Analysis of recombinant vaccinia virus-infected cell culture supernatant by sucrose density gradient centrifugation) As described in Reference Example 4, recombinant vaccinia virus v
XX39 1.2 × 10 ⁷ PFU / ml of human liver-derived C
1.0 ml (1.2 x
(10 ⁷ PFU), the virus was adsorbed to the cells at 37 ° C. for 2 hours, and then the cells were cultured with M-199 alone at 37 ° C. for 3 days.
200 ml of culture supernatant was centrifuged (3,000 xg, 5 '), and the supernatant was further centrifuged (48,000 xg, 14 hours, 4 hours).
℃), the precipitate was suspended in 2 ml of M / 75PBS, and sonicated (20 kHz, 200 W, 2 minutes).
Fractionation was performed by 0% (w / v) to 60% (w / v) sucrose density gradient centrifugation (160,000 × g, 15 hours, 4 ° C.). Each fraction was sample buffer [final concentration: glycerol 20% (v / v), Tris.HCl.
100 mM pH 6.8, SDS 2% (w / v),
2-mercaptoethanol 2% (v / v), BPB
0.02% (w / v)], heated at 100 ° C. for 5 minutes, and then subjected to 0.1% (w / v) SDS-12.5% polyacrylamide gel electrophoresis. Next, trans the gel
Each protein separated by electrophoresis was blotted to Hybond-ECL membrane (Ame
Blotting to Rsham (UK). Hy
The bond-ECL membrane contains 10 mM Tris.HCl pH 7.2, 150 containing 5% (w / v) skim milk.
mM NaCl, 0.05% (w / v) Tween-2
It is immersed in a solution of 0 (T-TBS) and incubated at room temperature for 1 hour for blocking. Then, anti-diluted 500 times with T-TBS buffer containing 1% skim milk.
37 core antigen monoclonal antibody (clone 29)
The reaction was carried out at 0 ° C for 1 hour, the Hybond-ECL membrane was washed twice with T-TBS containing 1% skim milk used for antibody dilution, and biotin-labeled anti-mouse IgG (manufactured by Kapel [Germany], diluted 500 times). ) Is reacted at room temperature for 1 hour. The Hybond-ECL membrane was washed twice, and HRPO-labeled streptavidin (Amersham [UK]) was used.
(Manufactured, diluted 500 times) at room temperature for 1 hour, and TT
Wash the Hybond-ECL membrane 4 times with BS and
L Western Blotting Detection System (Amers
Chem. Co., Ltd. (manufactured by UK), a chemiluminescence reaction is performed, the Hybond-ECL film is wrapped in Saran wrap, contacted with the X-ray film for 30 seconds, and then developed. As a result, NANBV core antigen activity and envelope antigen activity were observed at a sucrose concentration of 44 to 58% (w / v) (FIG. 7), which indicates that NANBV particles were obtained.

Continuation of front page (51) Int.Cl. ⁶ Identification number Office reference number FI technical display location C12P 21/02 C 9282-4B G01N 33/576 Z // (C12N 1/21 C12R 1:19) (C12P 21 / 02 C C12R 1:19) (31) Priority claim number Japanese Patent Application No. 3-132090 (32) Priority date Hei 3 (1991) May 8 (33) Priority claim country Japan (JP) (31) Priority Claiming number Japanese patent application No. 3-138493 (32) Priority date Hei 3 (1991) May 14 (33) Priority claiming country Japan (JP) (72) Inventor Chisato Mori 2-9 Hachiman-cho, Kanonji-shi, Kagawa No. 41 Incorporated Research Institute for Microbial Diseases, Osaka University Kannonji Research Institute (72) Inventor Akihisa Takamizawa 2-941 Hachimancho, Kanonji City, Kagawa Prefecture Kannonji Research Institute (72) ) Inventor Iwa Yoshida 2-941 Yawatacho, Kanonji-shi, Kagawa Prefecture, Osaka University Microbial Disease Research Group Kannonji Research Center

Claims (20)

[Claims] 1. An isolated non-A non-B hepatitis virus comprising at least one antigen selected from the group consisting of a non-A non-B hepatitis virus core antigen, a matrix antigen and an envelope antigen. particle. 2. The core antigen, the matrix antigen and the envelope antigen are 333 of the entire nucleotide sequence of the non-A non-B hepatitis virus of nucleotide sequences 1 to 9416 shown in FIGS. 2-1 to 2-16. The non-A non-B hepatitis virus particle according to claim 1, which is encoded by each of the nucleotide sequences of Nos. To 677, 678 to 905, and 906 to 1499. 3. The non-A non-B hepatitis virus particle according to claim 1, which has a ribonucleic acid corresponding to at least a partial region of the base sequence shown in FIGS. 2-1 to 2-16. 4. (a) at least 1000 non-A bases
1 prepared from non-hepatitis B virus genomic RNA fragment
Provided are 10 or less different cDNA clone groups each having a nucleotide number of 000 or more, wherein each cloned cDNA group of the cDNA clone group has the nucleotide sequence numbers shown in FIGS. 2-1 to 2-16 as a whole. 1st to 9th
It covers at least the region from the 333rd to the 5177th nucleotides of the non-A non-B hepatitis virus No. 416 whole nucleotide sequence, and (b) the nucleotide sequence obtained by sequential sequencing has at least the nucleotide number 333. CDNA fragment groups each having a base sequence previously determined so as to have a region corresponding to the region from No. 5177 to No. 5177
It is cut out from the A clone, and (c) the cDNA fragment groups each having these predetermined nucleotide sequences are sequentially ligated to each other, and the nucleotide sequences No. 1 to 9416 shown in FIGS. Non-A non-B hepatitis virus whole nucleotide sequence of at least nucleotide number 333 to 5177
Constructing a first deoxyribonucleic acid containing a nucleotide sequence
(D) a first deoxyribonucleic acid and at least one deoxyribonucleic acid obtained by replacing at least one base of the base sequence of the first deoxyribonucleic acid by degeneracy of the genetic code, A replicable recombinant DNA which is introduced into a replicable expression vector selected from a plasmid and an animal virus gene, and has a plasmid and at least one of the deoxyribonucleic acid introduced therein when the replicable expression vector is a plasmid. Or when the expression vector is an animal virus gene, a recombinant virus having the animal virus gene and at least one of the deoxyribonucleic acid introduced therein is obtained, and (e) the replicable virus used in step (d) is obtained. When the expression vector is a plasmid, the recombinant DNA is Or eukaryotic cells to form a transformant, and the transformant is then screened from a prokaryotic or eukaryotic cell culture and (f) the transformant obtained in step (e). To produce non-A non-B hepatitis virus particles by expressing the deoxyribonucleic acid, or by culturing the recombinant virus obtained in step (d) Producing a non-A non-B hepatitis virus particle by expressing an animal virus gene and said deoxyribonucleic acid in the form of a propagating recombinant virus comprising an animal virus and a non-A non-B hepatitis virus particle expressed therewith, And (g) the non-A
A method for producing non-A non-B hepatitis virus particles, which comprises isolating non-B hepatitis virus particles alone or in the form of the amplified recombinant virus. 5. The method according to claim 4, wherein the base sequence of the first deoxyribonucleic acid comprises the base sequence of base numbers 333 to 5918. 6. The method according to claim 4, wherein the base sequence of the first deoxyribonucleic acid comprises the base sequence of base numbers 333 to 6371. 7. The method according to claim 4, wherein the base sequence of the first deoxyribonucleic acid comprises the base sequences of base numbers 333 to 9362. 8. The method according to claim 4, wherein the base sequence of the first deoxyribonucleic acid comprises the base sequences of base numbers 1 to 9416. 9. A replicable expression vector selected from a plasmid and an animal virus gene and deoxyribonucleic acid, wherein the deoxyribonucleic acid is shown in FIGS. 2-1 to 2-1.
6, the first nucleotide sequence of the non-A non-B hepatitis virus Nos. 1 to 9416 and the first nucleotide sequence of the 333rd to 5177th nucleotides, and at least the first nucleotide sequence of 1 A recombinant which is selected from a second base sequence obtained by substituting one base by degeneracy of a gene codon. 10. The recombinant according to claim 9, wherein the first base sequence comprises a base sequence of base numbers 333 to 5918. 11. The recombinant according to claim 9, wherein the first base sequence comprises a base sequence of base numbers 333 to 6371. 12. The recombinant according to claim 9, wherein the first base sequence comprises the base sequence of base numbers 333 to 9362. 13. The recombinant according to claim 9, wherein the first base sequence comprises the base sequence of base numbers 1 to 9416. 14. A diagnostic agent for detecting non-A non-B hepatitis by an antigen-antibody reaction, which is used as a diagnostic agent.
13. A diagnostic agent comprising the non-A non-B hepatitis virus particle according to any one of claims 1 to 5 in an amount sufficient to exhibit an antigen-antibody reaction. 15. A non-A non-B hepatitis virus particle according to claim 1, 2 or 3 is contained in an amount sufficient to exert immunogenicity, and at least one pharmaceutically acceptable. Non-A non-B hepatitis vaccine, characterized in that it contains a carrier, diluent or excipient as defined above. 16. A non-A non-B hepatitis virus gene cDNA
Escherichia coli BK102 that possesses A
issue). 17. A non-A non-B hepatitis virus gene cDNA
E. coli BK106 that possesses A
issue). 18. The non-A non-B hepatitis virus gene cDNA
Escherichia coli BK112 that possesses A
issue). 19. The non-A non-B hepatitis virus gene cDNA
Escherichia coli BK146 that possesses A
issue). 20. Non-A non-B hepatitis virus gene cDNA
Escherichia coli BK147 that possesses A
issue).