JPH04169192A - Non-a non-b hepatitis virus antigen polypeptide, polynucleotide coding the antigen polypeptide and diagnosis of non-a non-b hepatitis using the same - Google Patents

Abstract

NEW MATERIAL:The non-A non-B hepatitis virus antigen polypeptide having the amino acid sequence of formula. USE:Vaccine for preventing and curing the infection with non-A non-B hepatitis virus. A reagent for detecting non-A non-B hepatitis virus antibody. PREPARATION:The objective polypeptide of formula can be produced e.g. by using the serum of a non-A non-B hepatitis patient as a starting raw material, concentrating and recovering the virus particle as a precipitate by polyethylene glycol precipitation process, extracting RNA from the virus by guanidine- thiocyanate process, etc., synthesizing a cDNA using the RNA as a template, inserting the cDNA into a phage vector to prepare a cDNA library, culturing the library, screening by enzyme immunoassay to select a positive plaque, extracting DNA from the plaque, treating with restriction enzymes, bonding to a vector, inserting into E.coli, etc., to effect the transformation of the cell and culturing the obtained transformant.

Description

DETAILED DESCRIPTION OF THE INVENTION (Industrial Field) - The present invention relates to non-A, non-B hepatitis virus antigens. More specifically, the present invention provides an antigen polypeptide that specifically exhibits an antigen-antibody reaction with non-A11 hepatitis B patient serum, a polynucleotide encoding the antigen polypeptide, and a method for treating non-A non-B hepatitis using the same. Regarding diagnostic methods.

(Prior art) Non-A, non-B hepatitis is a type of viral hepatitis that is thought to be caused by a transmissible virus, and the entity of which has been clarified is type ^ hepatitis virus (HAY>, B! !!
7 types of hepatitis virus (HBV), hepatitis D virus ()I
Virologically and serologically excluded hepatitis caused by DV), cytomegalovirus (CMV), Epstein-Barr virus (EBV), adenovirus, etc., which also infects liver cells and causes symptoms. It is.

Epidemiological evidence suggests that there are three types of non-A, non-B hepatitis: waterborne, which is prevalent in developing countries, post-transfusion (or needle accident), and developed countries. It is a sporadic (collectively acquired) type found in many countries. The waterborne type was thought to be caused by a different virus from the other two types, but in recent years, gene fragments of the main causative virus (hepatitis E virus) have been discovered by Gene!abs.
(USA) and the US r5CDC group [WO 89/12462.89/12641 pamphlet (1990), Reyes, G.R., e.
tal, 5science247, 1335 (199
0)13 In this specification, unless otherwise specified, "non-A, non-B hepatitis" does not include this water-borne type, but means the remaining post-transfusion type and sporadic type.

It is well known that post-transfusion hepatitis occurs in 10-20% of people who have received blood transfusions, but post-transfusion hepatitis
Alter [^1terJJ6. et al.
Annals of Internal Medicine
ine 77.691 (1972)], Prince [P
r1nce, ^, M,, et al, Lancet2,
241 (1974) Ko, Fine Stone [Fe1n
stone.

S, M, et al, New England
nd Journal of Medicine
292, 767 (1975)].

Later, the involvement of the hepatitis A virus was ruled out, and the diagnosis was ``non-A, non-B hepatitis.'' The fact that hepatitis is an infectious disease is that it can be caused not only by blood transfusions but also by blood products (coagulation factor preparations and immunoglobulin preparations), and depending on the loft of these preparations, hepatitis may or may not occur. It was estimated from an epidemiological study that there is. Filtration experiments, infection experiments after treatment with chloroform and formalin, and fractionation using density gradient centrifugation have revealed that this S-staining pathogenic factor is a small, enveloped RNA virus, probably Togavirus.
) was thought to be a species of

According to estimates by the Ministry of Health and Welfare (1988), more than 90% of post-transfusion acute hepatitis and about 50% of sporadic acute hepatitis are non-A, non-B hepatitis. Approximately half progress to chronic hepatitis, and 10 to 20 K progress to cirrhosis. Furthermore, it is thought that persistent infection with non-A, non-B hepatitis virus and/or the associated chronic inflammation are involved in the progression to liver cancer. Therefore, it is an urgent medical need to establish prevention and treatment methods for non-A, non-B hepatitis viruses.
As described below, even now, no reliable diagnostic, preventive, or therapeutic methods have been established.

Recently, two groups have reported that aX, a gene for a non-A, non-B hepatitis virus-specific antigen caused by blood transfusion. One is by the group of Chiron Corporation (USA) [Choo, Q-L, et at.

5science 244.359<1989), Ku
o,G,,et at,5science244,36
2 (1989), Special Publication No. 12-500880,
European Patent Application Publication No. 318216 (1989)
The other one was by Arima et al.'s group at Okayama University (currently Kagoshima University) [^rima, T., et al.
l, Hepatology 8.1275 (1988),
Arima, T., et al., Gastroen
tero-1ogia Japonica 24.68
5 (1989), Arima, T., et alGas
troenterologia Japonica 2
5.218 (1990), European Patent Application Publication No. 3630
25 Mei M31 <1990) Ko 1 Both antigens react specifically with the serum of many post-transfusion non-A, non-B chronic hepatitis patients and some sporadic non-A, non-B chronic hepatitis patients.
There was almost no reaction with normal human serum and serum from patients with acute hepatitis A and chronic hepatitis B. Furthermore, the genes encoding these were not derived from host cells, and these antigens were thought to be derived from non-A, non-B hepatitis virus genes.

The Chiron group named the virus containing this gene hepatitis C virus (HCV). Based on its genetic structure, HCV is considered to belong to the Flaviviridae family. However, infectious virus particles and infectious virus genomes have not been isolated, and the pathogenicity of this virus as a non-A, non-B hepatitis virus has not yet been proven.

Subsequently, several Japanese groups used immunoscreening methods and reverse transcription-polymerase chain reaction (CRT-PCR) methods from serum from patients with non-Non-B hepatitis.
A gene fragment with homology to HCV has been cloned. Compared to Chiron's HCV, these are approximately 5x~30% at the base sequence level and 5x~2x at the amino acid level.
A difference of approximately 0χ was observed, and it is thought that it originated from the Japanese variant of HCV1) [Maeno, M.
,et al,, Nucleic Ac1ds Re5
search1B, 26s5 (1990), Nobuo Takada et al., Liver 315upp1.217 (1990), Shuichi Kaneko et al., Liver It315upp1.213 (1990), Nobuyuki Kato et al., Liver 315upp1.214<1990)]
.

In this way, knowledge about non-non-A, non-B hepatitis viruses is accumulating; however, 1) subtype N of I'lCV and its distribution status, and 2) similar pathologies can be caused by blood transfusions other than HCV. 3) The "sporadic" non-A, non-B hepatitis that is prevalent in developed countries are all caused by the above-mentioned H-type hepatitis.
There has not been sufficient research on whether or not it is caused by CV; in other words, research has not been conducted on the identification of antigens necessary for the development of detailed and useful diagnostic methods and therapeutic amniotic membranes for non-A, non-B hepatitis. It is still not fully completed.

For example, some kits for measuring non-A, non-B hepatitis are already commercially available, but they only react with about 70% of serum from patients with non-A, non-B hepatitis after blood transfusion [Kuo , G., et alScience
244.362 (199p) Although the reaction rate with serum from patients with non-A, non-B hepatitis in the chronic stage is high, the reaction rate with serum from patients with acute-stage non-A, non-B hepatitis and serum from patients with sporadic non-A, non-B hepatitis is high. The reaction rate is low [Miya@ura, T, , et al.
al, Proc, Natl, Acad, Sci, U,
S.A. 87.983 (1990).

Kusiya Nishioka et al., Internal Medicine 64.1027 (1989),
Also, it takes an average of 4 months for antibody seroconversion [At1e
r, H, J, .

et il, N., Engl., J., Med., 30,14.
94 (1989)] and that the presence of the virus antibody does not necessarily correspond to viremia [Hiroaki Okamoto, Liver 1i
315upp1.40 (1990).

Koji Furusawa, Liver 1ia 315upp1.42 (1990
)], and there are still many problems to be solved in diagnostic methods for non-A11-B hepatitis virus antibodies.

(Problems to be Solved by the Invention) The present inventors have developed an antigen that is more useful than the conventional viral antigens, namely: 1) an antigen that has a high reaction rate to the serum of patients with non-A, non-B hepatitis after blood transfusion; 2) sporadic 3) An antigen that has a high reaction rate to the serum of patients with non-A non-B hepatitis; 3) An antigen that can also react with the serum of patients with acute non-A11-B hepatitis (an antigen that can detect viral antibodies that appear earlier in infection); 4) Strong correlation with viral blood swelling) As a result of intensive research to discover antigens that can detect viral antibodies, we obtained the gene for a new non-A, non-B hepatitis virus antigen from Japanese human plasma. When the base sequence of the gene was determined, it was found that there was homology with the previously reported base sequence (base sequence 5493rd to 5627th as shown in Figure 47 of Japanese Patent Application Publication No. 112-500880), but different base sequences were found. sequence, and was a novel non-A, non-B hepatitis virus antigen. In addition, the inventors completed the present invention after learning that this antigen is an antigen that can detect not only viral antibodies that appear in the chronic phase but also in the acute phase.

(Means for Solving the Problems) In carrying out the present invention, unless otherwise specified, molecular biology, microbiology, recombinant DNA,
and conventional techniques of immunology are employed. For such a method, please refer to the following experimental book.

Experiment Book 1: Sambrook, J., Fr1tsc
h, E. F., and Maniatis, T. Mo.
1ecular Cloning 2nd Ed
,; ColdSpring Harbor (
1989) Experiment book 2: DNA cloning 1
Volume (Glover, D, M, 1i) IRL Pr
ess, (1985) Experimental Book 3: DNA Cl
oning Volume 3 (Glover, D.M., ed.) IR
L Press, (1987) Experimental Book 4: H
andbook of experimental i
a++uno-1ogy, 4th ed, l~IV
Volume (Weir, D, M eds.), Black-well
(1986) Experimental Book 5: Current Protocols
in Molecular Biology (＾us
uber, F. M. et al., 1i) Joh
n Wiley & 5ons (1988) Experimental Book 6: Method in Enzymolo
gy Volume 185 (Goedd-e I, D, V, eds.) Academic Press (1990) (1)
Preparation of viral RN^ a) Preparation of RNA from patient blood HBs antigen negative, HBv-DNA negative, high ALT (
GPT) activity value (>351 tl/1) or non-A, non-B hepatitis patient serum were pooled and viral RN
Used as starting material for A1Fl production. Virus particles are concentrated and precipitated by polyethylene glycol precipitation method.
RNA is collected and extracted from the precipitate using a method such as the guanidine thiocyanate method [Chomczynski, P.
, and 5acchi, N,: Analytic
al Bioche+1stry 162.159
(1987)]. At this time, RNase
For example, heparin, polyvinyl sulfate, diethyl pyrocarbonate, vanadium complex, etc. can be added as an RNase inhibitor to prevent RNA degradation by.

In addition, chimpanzee serum infected with non-^non-B hepatitis virus can be used instead of human serum, but mutations may occur in the viral genes when the virus is passaged in animals of different species. When cloning antigens, human serum is preferably used as a starting material.

b) Preparation of RNA from patient liver tissue RNA was prepared using a standard method [Experimental Book Chapter 1.7] from liver tissue excised from a liver cancer patient who was thought to have been infected with non-^non-B hepatitis in the past. can be prepared.

(2) Construction of cDNA library Using the obtained RNA as a template and random hexamers as primers, use the Gubler-)1off++an method [G
ene, 25, 263 (1983)]. The cDNA is inserted into the λgtll phage vector.

The immunoscreening method using λgtll is a well-known method developed to isolate the antigen gene corresponding to a specific antibody [Young, R, ^, λn
dDavis, R.W., Proc.Natl, ^c
ad, sci, USA 80.1194 (1983),
Experiment book 2. p49-78], and in addition to λgtll, AZAP, λZAPII, pUc19. pUE
0 recombinant λ that can be inserted into vectors such as Xl
gttt infects the large intestine gA of the host (for example, strain Y1090), but under certain conditions it proliferates according to the bacteriophage propagation mechanism, and the previously integrated cDNA is also amplified accordingly. Under certain conditions, the bacteriophage lyses the host E. coli and forms a visually recognizable plaque.

(3) Immunoscreening of cDNA library using patient serum cDNA inserted into λgtll phage vector
Since λgtll is expressed as a fusion polypeptide with β-galactosidase (β-gal), screening a large number of recombinant λgtll with a specific antiserum raised against a specific antigen will yield a specific antigen polypeptide. A cDNA encoding a peptide can be identified by an immunochemical method.6 In the present invention, the CD produced in (2) above is used.
It is desirable to screen the IN^ library with one type of non-^non-B hepatitis hand serum or its pooled serum. Specifically, the fusion antigen protein expressed from a cultured dish with plaques, etc. Transfer to a nitrocellulose membrane, react with the patient serum described above as a primary antibody, react with peroxidase-labeled goat anti-(human IgG) IgG, etc. as a secondary antibody, and isolate plaques that show a positive color reaction. .

(4) cDNA DNA determination The base sequence of the antigen gene of the present invention thus obtained was determined by the Sanger method [Sang] on the recombinant λgtll DNA and on the plasmid vector DNA after subcloning.
er,F,,et al,,Proc,Natl,^c
ad, Sci, USA, , 74°5463
(1977), Experimental Book Chapter 1.13] can be used for determination using a commercially available sequencing kit.

(5) Method for detecting the virus genome in patient serum using the base sequence of the antigen gene A portion of 8 or more consecutive bases based on the disclosed portion of the base sequence of the non-A, non-B hepatitis virus polynucleotide of the present invention A polynucleotide comprising
ature 310, 105-111 (1984) 1, or a commercially available DNA synthesizer (e.g., Applied Biosystems (ABI), Model 38OA, DNA
It can be synthesized using tcPI, etc.). This polynucleotide hybridizes with the virus genome of the present invention and is used as a probe for identifying and detecting the virus.
or as a primer). When using the polynucleotide for diagnosis, it can be used as a labeled probe such as a release-resistant probe, a biotin fluorescent probe, or a chemiluminescent probe by a conventional method, but the above-mentioned hybridization cimin method or labeling method 5 can be used. The methods for amplifying and detecting signals are all known and can be carried out with reference to Toyozo Takahashi, DNA70-B, CMC (1990).

Until now, non-A, non-B hepatitis viruses exist in extremely small amounts in patient serum (1/1 of the hepatitis B virus).
0'li degree) is known. In such cases, 1 reverse transcription-PCR method [Ka
Wasaki, E. S., et al.
Proc, Natl.

Acad, Sc1. USA85.5698 (19
88), Hiroaki Okamoto, liver 31, 5upp1.40 (1
It is also possible to amplify and then detect the target virus genome using methods such as 990).

(6) Preparation of non-^non-B hepatitis virus antigen polypeptides and fusion antigen polypeptides a) Expression of recombinant antigen polypeptides and recombinant fusion antigen polypeptides using genetic recombination technology: The antigen polypeptides are known For example, expression in E. coli can be performed using the expression vector of Experiment 1, Chapter 17, Experiment 13,
p59-88, experimental book 6. For expression of pH-128 etc. in yeast, see Experiment Book 3, p141-161゜Experiment Book 6.
, P231-484, and the expression method when mammalian cells are used as hosts are described in Chapter 16 of Experimental Book 1, Experimental Book 3, p.
163-212, Experimental Book 6, p485-598, etc. 6 The expressed recombinant antigen polypeptide can be purified by protein purification techniques known in the art (@extrafiltration) from lysed cells or culture media. , centrifugation, dialysis,
Ion exchange chromatography, gel filtration, electrophoresis,
Affinity chromatography, HPLC, etc.; for example, Methods in Er+zymo-1og
Volume 7182 (Deutscher, M, P, @
) Acade+wjc Press (1990) etc.) can be used for purification.

Furthermore, as a simple method for expressing the fusion base polypeptide, for example, Escherichia coli Y1089 strain in which the recombinant λgtll clone Y15 of the present invention has been lysogenized is used as rPTG (1sop
ropyHhio-β-galactoside) [Jikken 2, p7 Rho 77] 0
The expressed fusion antigen polypeptide can be purified by gel filtration and/or immunoprecipitation using a mouse monoclonal antibody against β-gal (Boehringer Mannheim, 1083104) or affinity chromatography. Furthermore, expression of fusion antigen polypeptides using substances other than β-gaJ can be carried out with reference to Experimental Book 6, pages 129-165.

b> Preparation of the antigen polypeptide by peptide synthesis: The antigen polypeptide can also be provided by chemical synthesis based on the two amino acid sequences determined by the present invention. in this case,
It is preferable to synthesize the entire sequence into relatively easy-to-synthesize long fragments (10 to 10 residues) so that adjacent fragments overlap by about 5 to 10 residues. For detection, all of these split and synthesized peptide fragments are mixed and used, but once the antigenic region is identified by epitope mapping, only the fragments containing the antigenic region can be used alone or in combination. It can be used as

Various polypeptides containing the amino acid sequence of the antigen polypeptide can be synthesized manually by a liquid phase method or a solid phase method, or chemically synthesized by an automated synthesizer (for example, ABI's Model 430A peptide synthesizer). A polypeptide synthesized by a solid phase method according to the t-Boc method or the Fmoc method is excised from the resin using trifluoromethanesulfonic acid, hydrogen fluoride, trifluoroacetic acid, or the like. These synthetic peptides are
To further increase the degree of refinement.

Preparative reverse phase high performance liquid chromatography (HPLC)
It is also possible to purify by

(7) Method for detecting virus antibodies in patient serum by immunoassay The non-hepatitis B virus antigen polypeptide obtained by the present invention, or the antigen polypeptide consisting of a partial epitope thereof, can be detected using an immunoassay, for example. , non-A, non-B in biological samples, including body fluid samples such as blood, plasma, serum, cerebrospinal fluid, and human immunoglobulin preparations.
It can be used to detect the presence of hepatitis virus antibodies. In this immunoassay, the antigen polypeptide is contacted with the sample under conditions that allow the formation of an immunological bond (antigen-antibody conjugate) between the antigen polypeptide and the non-A, non-B hepatitis virus antibody in the sample. If even a small amount of antigen-antibody combination is formed, which is desirable, the presence of non-A, non-B hepatitis virus antibodies in the sample is known and can be detected and measured by appropriate means.

Such methods include immunoprecipitation, agglutination, radioimmunoassay (RIA), EIA, ELISA, Western blot assay, and binding immunoassay. Additionally, assay protocols using the antigenic polypeptides can be performed using competitive or non-competitive binding methods.

The antigen polypeptide may be labeled with a label depending on the assay method. Known labels that bind to the polypeptide can be used, such as fluorescent molecules, luminescent dye molecules, radioactive molecules, enzymes, covalent molecules, etc. Factors, biotin/avidin, colloidal gold, magnetic particles, etc. Furthermore, if the antigen polypeptide of the present invention is chemically modified, it can be bound to a carrier protein, carrier polypeptide, or a known support by known means. Examples of such supports include microtiter plates and beads made of polystyrene or polyvinyl, glass tubes and beads,
Also, romatographic supports such as paper, cellulose, cellulose derivatives, and silica can be used.

Among these assay methods, 'ij' is particularly useful for large-scale screening of patient serum, blood, or blood products.
ELISA, ring system method or Western blot assay etc. are suitable.

The simplest method is to express the antigen obtained according to the present invention as a fusion protein with, for example, β-gal, plot it on a nitrocellulose membrane, fix the antigen on a solid phase, and use the ELISA method to determine the non-A non-A. This is a method for detecting hepatitis B virus antibodies (such as immunoplaque assay and Western blot assay) (Example 4).

(Effects of the Invention) The antigen polypeptide of the present invention is useful for detecting non-A, non-B hepatitis virus antibodies that cannot be detected by conventional testing methods using antigens. Further, by using the non-A, non-B hepatitis virus polynucleotide sequence of the present invention, it is possible to detect virus particles (genomes) that cannot be measured with the detection sensitivity of conventional detection methods. Furthermore, using the antigen polypeptide, polyclonal antibodies and monoclonal antibodies that can be used for detection of the viral antigen and treatment of non-non-B hepatitis can be produced. In addition, the antigen polypeptide can be used in the production of vaccines for preventing viral infection and for post-infection treatment.

EXAMPLES The present invention will be explained in more detail with reference to Examples below, but these are not intended to limit the scope of the present invention.

Example 1: Extraction of plasma RN^ for cDNA^LT activity value is abnormally high (>701U/l>)
Frozen plasma 200% negative for antigen and HBV-DNA
After melting 0 ml at room temperature, fibrin was removed by centrifugation at 5000 rpm (Hitachi, l'1PR9-20-ter) for 20 minutes at 4°C. 80 g of polyethylene glycol 4000 was added to the supernatant and dissolved. After cooling on ice for 30 minutes, the mixture was centrifuged under the same conditions to collect the precipitate. RNA was extracted from the precipitate using the AGPC method using guanidine thiocyanate [Chomczyr+Ski,
P, and 5acchi, N, :^nalyti
cal Biochemistry, lJd, 15
6 (1987)]. That is, double the amount of the precipitate (1 g of precipitate was replaced with 11X) in solution 6D (6) 4 guanidine thiocyanate, 25 mM sodium citrate pH 7.0, 0.5% Sarcosyl, 0.
After dissolving in 1M 2-mercaptoethanol), add 0.1 volume of 2M sodium acetate (pi(4',O),
Then equal volumes of phenol and 0.2 volumes of chloroform:
Extraction was performed with isoamyl alcohol mixture M (49:l). After cooling with water for 15 minutes, the mixture was centrifuged, the upper layer was collected, and an equal amount of 2-propatool was added to precipitate RNA. After centrifugation, sedimentation! i thing 05 evening 9 melt? ! ! 4T) (
After dissolving in 4@guanidine thiocyanate, 25+mM sodium citrate pH 7.0; 0.5% Sarcosyl, 0.1M 2-mercaptoethanol),
Double volume of ethanol was added to precipitate RNA again. After centrifugation, the injected material was washed with 75% ethanol and dried. The precipitate was dissolved in 50μ9 sterile distilled water and RN
Solution A was used.

Example 2: Creation of cDNA library cDNA synthesis was performed using 10 μl of RNA solution as a starting material. R
Double-stranded cDNA can be created from NA using the commercially available cDNA synthesis system plus (Amajam, RPN-1256).
Z) was used. The conversion rate from RNA to single-stranded cDNA was about 2%, and the conversion rate from single-stranded cDNA to double-stranded cDNA was about 100%.

The synthesized double-stranded cDNA was precipitated with ethanol and then precipitated with 100. u II in TE solution (10mM Tr
is-Cff.

Commercially available cDN^ synthesis kit (Pharmacia IJB, 27-
9260-01> was used to carry out the following reaction. c.
Add the DNA solution to the 5ephacryl that comes with the kit.
By passing it through an S-300 span column! ×T4
Next, the ligase solution was subjected to buffer exchange, and then an EcoRI adapter addition reaction and a T4-kinase reaction were performed according to the instructions.

The adapter that did not bind to the cDNA was 5ephac
The cDNA in the eluate, which was removed using a rylS-300 span column, was ethanol precipitated with 5 μg of carrier tRN^. The precipitate was washed with 75% ethanol and then air-dried.

Next, use the commercially available cDNA cloning system λgtl
・Adapter method (Amajam+, RPN 1280)
The following reaction was carried out using After adding 5μ9 of sterile distilled water, 4μ9 (2μg) of λgtll EcoR1 arm, and 1μ9 of L/X buffer to the precipitate and dissolving it, lμQ (2,50) of T4 ligase was added.
After leaving it at 15°C overnight, GIGAPACK n
Goldλ DNA packaging kit (Str4t
The above reaction product (containing recombinant λgtll DNA) was in v.
An intro packaging reaction was performed to create a cDNA library (Lot, B).

Example 3: Cloning of non-A, non-B hepatitis virus antigen genes by immunoscreening The reaction between plaques in the cDNA library (Lot, B) and primary and secondary antibodies was performed using the commercially available SuperScreen immunoscreening system. (Amajam, RPN 1
It was carried out according to the method of 281Z). The primary antibody used for screening was pooled serum from non-A, non-B hepatitis patients (
5 patients with convalescent hepatitis and 5 with chronic hepatitis) were absorbed with Escherichia coli lysate (Bio-Rat, 17-32 (15) (21g lysate/19 serum)) and mixed with solution A (10mM Tris-CQ, pH 7). ,515
It was diluted 20 times with 0mM NACl, 2% (W/V) BSA) and used. For the secondary antibody, peroxidase-labeled goat anti-(human IgG) IgG (Kappel, 3201-0081) was diluted 500 times. The color reaction was performed using the commercially available Lambda Lift Detection Kit GA.
R-HRP (Bio Rat, 17-3556)
This was done using

Ten positive plaques (colored blue-purple on a nitrocellulose filter) were isolated from approximately 500,000 plaques. One clone among them was named Y15, and the following analysis was continued.

Example 4: Antigen specificity test of clone Y15 a) Immunoplaque assay A mixture of Y15 clone and negative clone at a ratio of 2=8 was plated, and using the above-mentioned immunoscreening method, non-A, non-B Serum from chronic hepatitis patients (with history of blood transfusion) (5 patients), non-A, non-B acute hepatitis patients (blood transfusion!!
! No) serum (6 people), non-A, non-B chronic hepatitis patient (with history of blood transfusion) serum (5 people), normal human control serum (5 people>
The reactivity with serum from patients with chronic hepatitis B (5 patients) and blood from a patient with acute hepatitis A (1 patient) was investigated. As a result, clone Y
15 was shown to react positively only with non-A, non-B hepatitis patient serum (Table 1).

Patients with non-A, non-B acute hepatitis 5 315 Non-A, non-B!
! 2 Acute hepatitis No blood transfusion 6 2/6 Non-A, non-B chronic hepatitis No blood transfusion 5 415A acute hepatitis
1 0/Type IB chronic hepatitis 5 0
15 Con Law 1. 15 That is, it was V& recognized that clone Y15 encodes an antigen (epitope) that is immunologically recognized by serum derived from non-A, non-B hepatitis patients. It was also found that this antigen reacts with serum from patients with acute non-A, non-B hepatitis.

b) Western blot assay λgll (parent strain) lysogen and recombinant λgtllY1
5. Preparation of lysogenic bacteria, cultivation of the above lysogenic bacteria, and induction of expression of β-gil and β-gal fusion antigen polypeptides were carried out according to the method in Experimental Book 3, page 76. 6. After expression induction, the culture solution was The protein concentration was determined by sonication and the Bio-Rad method (Bio-Rad method).
Rat, 500-0001). A protein amount equivalent to 5 μg from each disruption solution was applied to 5DS-F'AGH,
After electrophoresis, use a semi-dry electroplotter (Integ
ated 5eparati.

A tan plot for each sea urchin was performed using a computer system (N Systems). The various sera shown in Table 1 were used as primary antibodies, and the reactivity of the various sera to β-gal and β-gal@combinant antigens was examined using the immunoscreening method described in Example 3). As a result, a color reaction was observed only in the combination of β-gal fusion antigen polypeptide (approximately 100 kd) and non-A, non-B hepatitis patient serum (the positive rate was the same as the results in Table 1). Antigen specificity was confirmed.

Example 5: Determination of the base sequence of cDNA derived from clone Y15 and the antigen polypeptide sequence encoded within the sequence Clone Y15 was grown by the plate lysate method described in 2.118 of Experimental Book 1, and then phage DNA was obtained. was prepared. After 5 μg of phage DNA was digested with EcoRr, it was purified by phenol extraction and ethanol precipitation.

After treating 3 μg of EcoRI digest with alkaline phosphatase, 5' end labeling was performed using T4 kinase and γ)2P-ATP. After purifying the DNA fragments by phenol extraction and ethanol precipitation, 5. O
x-polyacrylamide gel electrophoresis and autoradiography to examine the length of the cDNA excised by EcoRI digestion. As a result, the size of cDN^ was found to be approximately 130 base pairs.

Next, the above-mentioned EcoRI digest and the dedigested and stopped phosphorylated Z19 after EcoRI digestion (7yl Schiff LKB,
27-4986-01) was transfected into JM109 (Takara Shuzo, 9052), and a plasmid containing an insert P of about 130 bases was isolated (named pTZ19-Y15).

The base sequence of the cDNA incorporated into pTZ19-Y15 is DyeDeoxy Terminator Ta.
q Sequencing Xit (＾Bl.

401070) to perform a sea-fence reaction,
It was determined by electrophoresis using a DNA sequencer (8Bl, 373^ type).

To decide) 413 ply 7-M4 (Takara Shuzo, 3832)
Both strands were read and determined using a 1413 Brimer RV (Takara Shuzo, 3830).

In addition, in order to confirm which chain of the determined sequence was involved in forming a fusion protein with β-gal protein on recombinant λgtll, we used λgtll EcoR1 site primers forward and reverse (New England Biol).
abs, 1218.1222)
Base sequencing was performed using A directly as a template. Based on these results, the nucleotide sequence encoding the antigen that specifically reacts with non-A, non-B hepatitis patient serum was determined as shown in Figure 1.
In addition, the amino acid sequence of the antigen was determined as shown in Section 2.

[Brief explanation of drawings]

FIG. 1 shows the nucleotide sequence encoding the non-A, non-B hepatitis virus antigen polypeptide of the present invention. FIG. 2 shows the amino acid sequence of the non-A, non-B hepatitis virus antigen polypeptide of the present invention. Patent applicant Masaru Ariuma Yamanouchi Pharmaceutical Co., Ltd.

Claims (10)

[Claims] (1) Non-A, non-B hepatitis virus antigen polypeptide shown by the amino acid sequence below [gene sequence is available]. ] (2) A chemically synthesized antigen polypeptide according to claim (1) or a group of polypeptide fragments containing the amino acid sequence thereof (3) A fusion antigen polypeptide having the amino acid sequence of claim (1) (4) The antigen polypeptide is β-galactosidase (β-
The fusion antigen polypeptide according to claim (3), which is fused with (gal) (5) Recombinant λgtll clone Y15 expressing the antigen polypeptide according to claim (4) (6) A polynucleotide encoding the antigen polypeptide according to any one of claims (1), (3), or (4). (7) Non-A, non-B hepatitis virus polynucleotide shown by the base sequence below [gene sequence is available]. ] (8) A polynucleotide probe for detecting a polynucleotide derived from a non-A, non-B hepatitis virus, the polynucleotide probe having a length of 8 consecutive bases or longer according to claim (7). (9) A host cell transformed with a recombinant expression vector to produce the antigen polypeptide according to any one of claims (1), (3), or (4) is used to express the polypeptide. A method for producing an antigen polypeptide, which comprises culturing under conditions of (10) Claim (1), (2), (3) or (4)
Non-A, non-B hepatitis in a biological sample by measuring the immunological conjugate formed by the reaction between the antigen polypeptide described above and non-A, non-B hepatitis virus antibodies present in the biological sample. Method for detecting non-A, non-B hepatitis virus antibodies to confirm the presence of virus antibodies