JPH01124387A - Manifestation vector having dna coding non-a non-b hepatitis specific antigen, transformant and production of said antigen - Google Patents

Abstract

PURPOSE:To produce a non-A non-B hepatitis specific antigen, by introducing a DNA-containing DNA fragment coding a non-A non-B hepatitis specific antigen in a cloning site existing at the downstream side of a promoter, thereby forming a manifestation vector. CONSTITUTION:A DNA fragment containing a DNA coding a non-A non-B hepatitis specific antigen is introduced into a cloning site existing at the downstream side of a promoter of manifestation vector. The obtained manifestation vector containing the DNA fragment is introduced into a host to effect the transformation of the host and the resultant transformant is cultured. The non-A non-B hepatitis specific antigen produced and accumulated in the cultured product is separated therefrom A large amount of non-A non-B hepatitis specific antigen protein can be produced by this process at a low cost.

Description

DETAILED DESCRIPTION OF THE INVENTION <Industrial Application Field> The present invention is directed to the production of non-A, non-B in a host by recombinant DNA technology.
DN encoding an antigen specifically seen at the onset of type hepatitis
The present invention relates to an expression vector containing A, a transformant transformed with the vector, and a method for culturing the transformant to produce a non-A, non-B hepatitis-specific antigen.

<Problems to be Solved by the Prior Art and the Invention> Among viral hepatitis, viruses of type A and type B have been discovered, and diagnosis by immunological methods has become possible.

However, so-called non-A, non-B hepatitis, which is neither type A nor type B, is said to account for more than 90% of post-transfusion hepatitis, and is clinically reported in Japan.

as, 2721I, (/q7ri); , T, Bi
ol, Mea, (Journal of Biological Medicine), 19°, 2 evenings, y, (lqqg,)
), but the causative virus has not yet been identified, and it has only been confirmed that human non-A, non-B hepatitis can be transmitted to chimpanzees. , qt3. (
tqqg)').

Many researchers have searched for antigen-antibody systems related to non-A, non-B hepatitis, focusing on patient serum, but no clear system has yet been found. Therefore, the diagnosis of non-A, non-B hepatitis is based on hepatitis A, hepatitis B, and even known viruses that are known to cause liver damage, such as C!
MV.

Currently, the so-called exclusion diagnosis method is used to diagnose whether or not hepatitis is caused by H3V, EBV, etc., and if it is not applicable, to diagnose non-A, non-B hepatitis, which is time-consuming.

Tokukai Showa/-77Ag! rla publication and the same AI-&&
Publication No. 94 proposes the purification of non-A, non-B antigen proteins from human and chimpanzee hepatocytes that are useful for the direct diagnosis of non-A, non-B hepatitis, and monoclonal antibodies that are useful for the treatment thereof. .

However, when used as a diagnostic reagent, for example, a large amount of non-A, non-B hepatitis-specific antigen protein is required; Purification is not necessarily a suitable method.

Means for Solving the Problems> Therefore, the present inventors have conducted extensive studies to produce the antigen protein in large quantities using genetic engineering techniques, and have found that non-A, non-B For the first time, we have isolated and obtained a gene encoding a hepatitis-specific antigen protein, obtained an expression vector containing the gene, and completed the present invention.

In other words, the gist of the present invention is to use a DNA fragment containing a DNA fragment encoding an antigen specifically observed at the onset of non-A, non-B hepatitis, or a non-A, non-B hepatitis-specific antigen having similar physiological activity to a promoter. An expression vector characterized by being introduced into a downstream cloning site,
The present invention relates to a transformant obtained by transformation with the expression vector, and a method of culturing the transformant to produce a non-A, non-B hepatitis-specific antigen.

The present invention will be explained in detail below.

The DNA fragment used in the present invention, which contains a DNA encoding an antigen specifically observed at the onset of non-A, non-B hepatitis, is prepared by the following method.

First, the liver tissue of a human or chimpanzee individual who develops non-A, non-B hepatitis (in the present invention, includes individuals who develop so-called hepatitis, which has been named in recent years) is homogenized in an aqueous solution of guanidium thiocyanate, etc., and then Chirgwin et al.'s method (Biochemistry)
King L! ;291I-3299.

(/q7q)), total RNA is separated as a precipitate by cesium chloride equilibrium density gradient ultracentrifugation. After separation, total RNA was extracted by phenol extraction and ethanol precipitation.
refine.

Since the mRNA of the antigen gene generally contains the po17 A portion, it is converted into oligo(dT) using a conventional method.
It is purified by cellulose column chromatography to isolate poly(A)-containing RNA (poly A+RNA), which is used as an mRNA raw material. Using this mRNA raw material, random primer method (Yousuke Ebina et al., Ce
11 (cell), 2, 75g of Riq.

(/9go)] to obtain a cDNA library for these p017 A+RNAs. For example, using an arbitrary primer as large as O base and using reverse transcriptase, the above-mentioned mRNA is
Randomly synthesize cDNA for A. Furthermore, this cDNA is methylated with a DNA methylase (e.g. EcoR emeterase) to protect the restriction enzyme cleavage site present in the cDNA, and then a DNA linker containing the restriction enzyme cleavage site at both ends [e.g.
ATTCG)] and the restriction enzyme (e.g. KcoR
Digestion is carried out according to I).

This product is cloned into a cloning vector such as a plasmid or λ phage.

For example, the expression cloning vector λgt/t
D N A (: Young, R, A et al., Pro.
Natl, Acad.

SC1, U, S, A (Proceeding National Academy of Sciences ty, s, A,), r
z.

//914-179g, (19g3)].

Such λg, t // cDNA integrated into the phage
Since A is integrated into the β-gal gene on the λg, t // phage, the infection of the 7 age to E. coli is eliminated.
Expression as a fusion protein with β-galactosidase is easily confirmed by induction of the lactose operon promoter on the phage by addition of a substance such as G (inglovir-β-D-galactopyranoside).

In this way, the cDNA-incorporated λgt//phage was infected with Escherichia coli using the method of Tomizawa et al. (1-Bacteriophage Experimental Methods, pages 99 to 1tlI, published by Iwanami Shoten 7970, Yuzuki 30). Culture in a medium containing IPTG etc. By selecting the formed plaques using a non-A, non-B hepatitis-specific monoclonal antibody and using methods such as immunoscreening, it is easy to obtain the desired cD.
NA can be obtained.

The antibodies used in this immunoscreening method are disclosed in Japanese Patent Application Laid-open No. 1.
/-771, gjA publication, or the same AI-! ;A/?
! It can be prepared according to the method described in . The screening method may also be performed by the Western blotting method described in these publications.

Furthermore, phages were propagated from the positive immunoscreening plaques by the method of Tomizawa et al., and T and M
Aniatis et al.'s method (Molecular Clone
ing (molecular cloning).

Co1d Spring Harbor') (La
borator y PP t! r(/9t2)), and after cutting with an appropriate restriction enzyme such as KcoRI, the DNA was purified using the method of Maxam and Gilbert (
Methoas 1n KnZ7m010g7 (Methoas in Enzymology), t, s, tI99
-Taotθ, (79KO)) or with restriction enzymes, and further cloned into M/3 phage and Sanger
et al.'s dioxy method (Proc, NatL Acad,
Sci.

U, S, A, (Proceedings of the National Academy of Sciences U, S, A,), 2is
tat, 3. (tqqq) The base sequence of the target cDNA segment can be determined.

In this way, a cDNA encoding a non-A, non-B hepatitis-specific antigen
A fragment is obtained. However, the DNA fragment thus obtained is usually a partial cDNA fragment of a gene encoding a non-A, non-B hepatitis-specific antigen. The full-length cDNA was converted to poly A+- in the same manner as above.
mRNA was isolated and purified, and Okayama-Berg
vector, Breimer's method (Molecula
r and CrllularBio10g7 (Molecular and Cellular Biology), Main, /l
, /-/70. (/9g co)) to obtain a cDNA library. The cDNA-containing plasmid thus prepared was subjected to a conventional method, for example, the method of Hanahan (J.
Mo1. :5to1. (Journal Op Molecular Biology).

E. coli etc. are transformed using the following method (lqg3)) to obtain an ampicillin-resistant strain. This transformant is screened by colony hybridization method or the like using the above DNA fragment as a probe.

Preferred methods for producing such probes include the streptavidin method and the nick translation method using photobiotin nucleic acid and 32P nucleic acid.

Colonies containing cDNA clones obtained in this way were cultured, and the method of Birmboim et al. (Nucleic
Ac1d Res, (-'-Nikreitsk Azid Research),? , /st3. (/q7q)]
Obtain plasmid DNA according to the following procedure, cut it with an appropriate restriction enzyme, and then clone it into M/J phage or plasmid pVo/2, etc. by the method of Maxam and G11bert described above or after cutting with a restriction enzyme,
The nucleotide sequence of the full-length cDNA segment of interest is determined by the above-mentioned dideoxy method of Sanger et al.

FIG. 7 shows the base sequence of the DNA encoding the non-A, non-B hepatitis-specific antigen.

Of course, the DNA fragments used in the present invention are not necessarily required to have the same base sequence;
The substance encoded by the DNA contained in the A fragment is
As long as it encodes a substance that has the same physiological activity as the non-A, non-B hepatitis-specific antigen, it may be a base sequence in which part of the base sequence is replaced or deleted, or a base is added.

The expression vector of the present invention contains a promoter at a position capable of transcriptionally controlling the DNA encoding the non-A, non-B hepatitis-specific antigen obtained as described above.

Any promoter may be used as long as it can be expressed in the host, but preferably one that can be controlled.

For example, when using microorganisms such as E. coli and Bacillus subtilis as hosts,
The expression vector contains a promoter, a ribosome binding sequence,
It preferably consists of a non-A, non-B hepatitis-specific antigen gene, a transcription termination factor, and a gene that controls a promoter.

Examples of promoters include promoters derived from Escherichia coli, phage, etc., such as tryptophan synthase opemen (trp
), lactose operon (1ac), lipoprotein (
lpp), recA, lambda phage PL+ PR
%T5 early gene "251"26 promoter and the like. These may be created by chemical synthesis. Also, tac (trp:lac),
A hybrid promoter such as trc (trp:lac) or Pac (phage: Escherichia coli) as shown in FIG. 2 may be used.

The liposome-binding sequence may be derived from Escherichia coli, phage, etc. Synthetically created hepatitis-specific antibody genes may be used as they are;
) July, 131. -13.9, (/91
) ), etc., resulting in extra base sequences (non-coding
It is preferable to exclude the area).

Although the transcription termination factor is not necessarily required, it is preferable to have one that is ρ-independent, such as II) 1) terminator-1 trp operon terminator, liposome RN'A gene terminator, and the like.

Further, as the expression vector, a normal plasmid may be used, but a plasmid that has a high copy number in E. coli or Bacillus subtilis, such as pBR, etc. , 2.2 system plasmid, pUB/
/(7 series It is preferable to use a plasmid etc.

Furthermore, the sequence order of the factors necessary for these expressions on the expression plasmid is from the 5' upstream: the promoter, the SD sequence, the non-A, non-B hepatitis-specific antigen gene, the structural gene, and the transcription termination factor. desirable. Furthermore, the order of repressor genes, marker genes (drug resistance, etc.), plasmid replication initiation sites, etc. necessary for controlling transcription is not particularly limited.

As a host transformation method, Molecu for Escherichia coli is used.
lar Clothing (Molecular Cloning), 2! ;, 0-2! ;, 3. (/9g2), and for Bacillus subtilis, Molec, Gen, Gene
t, (Molecular General Genetics),
ybg, /ls-//! , (/9'/9) and Proc
, Nat, Acad, Sci, U, S, A.

(Proceedings of the National Academy of Sciences U, S, A, ), 3, 1072-
107g.

Conventional methods such as those described in (/qsg) can be used.

As a method for culturing the transformant, a medium (Molecu'larC1o
ning (Molecular Croni/G).

t,,g-73,(/qq2)) can be used.

In addition, the culture temperature may be 2g-Dako℃, but it is preferably within a range that does not induce the expression of heat-shock proteins etc.
It is preferable to carry out the reaction at a temperature of y to 30°C.

To purify the target product from the host, for example, host cells are disrupted using lysozyme-surfactant or ultrasound, and then the insoluble fraction (non-A, non-B hepatitis-specific antigen is fractions) were collected by centrifugation, and after converting them with a surfactant (e.g. 0.07% SDS, etc.), monoclonal antibodies (JP-A-47-54/Q4
Publication No. 4/-/7Ag! It is easily purified by column chromatography using RB Publication).

In eukaryotic cells, such as animal cells, the following are preferred.

Promoters include 5vao early promoter, 5vao early promoter,
vlIO late promoter, apoliboprotein E gene promoter, apoliboprotein A-■ gene promoter, heat shock gene promoter (Pr
oc, Natl, Acad, Sci.

U, S, A, (Grosseeding National Academy-Op Science U, S, A,) , □,
703g-qotta, (tqgl)), metallothionein gene promoter (Proc, Natl, A
cad, Sci, U, S, A, .

And A! ;//-1,! /j, (79gO)), H8
VTK promoter, adenovirus promoter (
Ad2 major late promoter (Ad 2MLP promoter)), retroviral LTR (LongTerm
Inal Repeat), etc., but Sv Ko0 promoter and metallothionein gene promoter are preferred.

The expression vector contains the y splice site (5'θplicej
(unction donor 5ite), intron and 3' splice site (3' 5plice jun
splice sequence DNA (exon-intron junction site,
A common base sequence has been found around the same junction site,
The so-called GT/AG rule holds that an intron region always begins with two bases of GT (one donor site) and ends with two bases of AG (one acceptor site).

Seven or more such splice sequence DNAs may be present in the expression vector, and their positions may be upstream or downstream of the non-A, non-B hepatitis specific antigen structural gene.

A specific example of the above splice sequence DNA is rabbit β
- Exon co and exon 3 of the globin gene (5c
(Science), 24,339゜(/9Wata)), metallothionein gene promoter,
Mouse metallothionein-■ gene (Proc,
Natl, Acad-Sci, U, S, A.

(Proceeding National Academy of Sciences U, S, A, N9.Zneg,t,s1.y,
(see /qgo)) splice sequence DNA present in
can be mentioned. Furthermore, the y and 3' splice sites do not need to be derived from the same gene; for example, a sequence in which the 5' splice site contained in adenovirus DNA and the 3' splice site derived from the Ig variable region gene can be used. .

The expression vector of the invention further contains a polyadenylation site. The polyadenylation site is present downstream of the non-A, non-B hepatitis specific antigen structural gene. Specific examples of polyadenylation sites include those derived from SV'IO DNA, the β-globin gene, or the metallothionein gene.

t, the polyadenylation site of the β-globin gene and 5
VI) DNA polyadenylation sites may be linked.

The expression vector of the present invention may have a dominant selection marker that allows selection of transformants. Even without a selection marker in the expression vector, the double transformation method (cotr
Transformed animal cells of the present invention can be selected by (transformation).

Such selection markers include the DHPR gene that confers MTX (mentrexate) resistance, the construct gene of Herpes simplex virus (H8V), which allows selection of tk-strains transformed underground in HAT medium, and the 3'- Examples include the aminoglycoside 3'-phosphotransferase gene from the Inuyoga transposon Tnj that confers resistance to the deoxystreptamine antibiotic Gll/g, the bovine papillomavirus gene that enables morphological differentiation through multilayer growth, and the aprt gene. .

In addition, in order to select animal cells transformed with the expression vector of the present invention by the double transformation method, a plasmid or other DNA containing the above-mentioned selection marker gene may be used.
can be transformed together with an expression vector, and transformed cells can be selected based on the above-mentioned phenotypic traits resulting from the expression of a selection marker.

It is advantageous for the expression vector to contain a plasmid fragment having an origin of replication derived from a bacterium such as E. coli, as this allows cloning in the bacterium.

Such plasmids include pBRJ 22, pBR
3J? , pML, etc.

Specific examples of plasmid vectors used as expression vectors include the 5vtto early promoter, the rabbit β-
Splice sequence DNA derived from the globin gene, polyadenylation site from the rabbit β-globin gene, s
Polyadenylation sites from the vg o early region as well as pB
pKOR (Proc, Natl, Aca
d, Sci, U, S, A, (Proceedings
National Academy of Sciences U, S, A,
), 7 g, 15.2 g, (79 g/)),
pKORH2 (Nature, y
oq.

pBPV MT/ (Proc, Na
tl.

Acad, Sci, U, S, A, , go, 3
qg, (/qr, y)), etc.

Animal cells transformed with expression vectors include OH
Examples include O cells, COS cells, mouse L cells, mouse 0727 cells, and mouse 0727 cells.

The expression vector of the present invention can be introduced into animal cells by a transfection method, a microinjection method, etc. The transfection method includes Ca-P
O4 (virology), b,
5b-1Ibq, (tqq3)) is the most common.

Animal cells transformed by transfection can be cultured in a suspension medium or a fixed medium by conventional methods.

As a culture medium, MKMSRPM/&'IO etc. are commonly used.

The produced protein can be separated and purified in the same manner as when produced by microorganisms.

(Example) The present invention will be explained in more detail with the following Examples, but the present invention is not limited by the following Examples unless it exceeds the gist thereof.Reference Example/ Non-A, Non-B type Preparation of poly(A) RNA from the liver of a hepatitis-infected chimpanzee using the guanidine thiocyanate-lithium chloride method [Cathala et al.
knee), 2. ．． 329. (/9za3)] according to Poly(A
) was prepared as follows.

Infected liver syringe from chimpanzees infected with non-A, non-B hepatitis
was removed and immediately frozen in liquid nitrogen. Put this in a Waring blender with liquid nitrogen and add 3.00
It was ground at 0 r, p, m for 2 minutes. Add this to 5M guanidine thiocyanate, /'OmMKDTA,
SOmM) Lis-HCl (pH 7) and g%
(V/V) Solution 7 consisting of β-mercaptoethanol
Teflon homogenizer (5r, p, m-
) and solubilized.

This solubilized material 20- is placed in a centrifuge tube for 5,7 M0s.
Gently place it on the O1 solution 70- and add HitachiRPS.
At 2g-20-tar, 27.00Or, p, m, 2
After centrifugation for 0 hours, RNA was collected as a precipitate. This RNA
Precipitate with 0.7% sodium lauryl sulfate, /mM
EDTA, 10rnM) Lis-HC
1 (pH 7,!;) /θ-, extracted with phenol-chloroform, and recovered by ethanol precipitation. The RNA obtained was approximately J,9! r■/(7mM) Risu-HC! 1 (pHg, 0) and /rnt in a solution consisting of mMEDTA. 4
Incubate for 5 minutes at 5°C. /- of! ; MNa
Added Ol. The mixture was transferred to an oligo[dt]cellulose e-column [PL BioChemical Co., Ltd.
Chemical) chromatography (column volume θ, 1rnt). Adsorbed Po1,1 (A
) /θmM trisac1 (pH 7
, 5) and /mMFliDTA to obtain about 100 μl of mRNA containing poly(A).

First, poly(A) mRNA / 0 μg was added to RT buffer (
20mM Tris-HCl (pHg, g), θ,
/MKCI, 72mM MgC1□, 2mM Mn
C1□)! ; Dissolve in 0 μL and random primer d(N)6 [BL Biochemical (P-L
1 gttli (manufactured by Biochemical) was added and denatured by heating at 95° C. for 3 minutes. This was slowly cooled to room temperature and the random primer was annealed. To this / 'OmM4<NTP / 0 μL, reverse transcriptase-co! ru (manufactured by Takara Shuzo Co., Ltd.) was added, water was added to make a total of 100 μt, and the mixture was reacted at 92° C. for 7 hours.

Using 50 μt of the above reaction solution / OmMNAD μt1
/ OmMIIdNTP / 01tL, RNas
e Hju, E. coli ligase/u, E. coli DNA polymerase ■6, Ju,/θ times the concentration of T-Hong DNA ligase buffer (0,/M Tris-HCl
(pH7,5) '0. / MDTT.

& OmMMgO1□) / Otit, and total /
00 lit system, reacted at 37°C for 7 hours, and
Double-stranded DNA was synthesized.

The double-stranded DNA obtained as described above was extracted with the same amount of water-saturated phenol, and after removing the phenol in the aqueous layer with ether, ethanol precipitation was performed.

The obtained precipitate was dissolved in 50 μt of water, and 10 times the concentration of T was added.
11 DNA polymerase buffer (o, 3.?M Tris acetate (pH 7,9), 0.6 A M potassium acetate,
0.7M magnesium acetate, 3mM DTT)/0
μt.

Add /OmM4ZdNTD/0ttL and 6u of TQ DNA polymerase to make /001tt system, 37
The reaction was carried out at °C/hour to obtain DNA with blunt ends of the -heavy chain. This was extracted with phenol as described above,
After protein removal, DNA was purified by ethanol precipitation and air-dried.

To this one! ; OmM) Lis-Hc1 (pHq, s
)/mMNa, KDTAX&mMDTT, 20
ttL, 100 uMe-adenosyl-L-methionine λμt, i, g■/Scallion mco R emethylase θ
・Add -μt, react at 37°C for 7S, and remove DNA.
The EcoRI restriction enzyme cleavage site on the fragment was methylated, and then heat treatment was performed at 70°C for 75 minutes to inactivate the enzyme.

Next, the 3′-phosphorylated Eco RX linker (GG
AATTCC) was added in an amount 100 times the number of NA molecules for total synthesis, and 70 times the concentration of T+DNA IJ gauze buffer (0,!; M Tris-HoI (pH 7,!r) was added.
, 1, OmM MgO1□, / OmMDTT) to 3
Add μ, 0, i MATP! rttL,TIDN4
5 u of ligase was added to make a total system of 50 μt, and after reacting for 1° C./A hour, the enzyme was deactivated by heating at 70° C. for 70 minutes. Then add 10x concentrated Eco RI buffer (/s
M, ) Lis-HCI (pH 7,5),
0. k M Na1l, 40 m
MMgC12) at 10ttL, F2coRT, 10θ
U was added to make a system with a total of 1θOμt, and the mixture was reacted at 37°C for 2 hours, and the excess linker was excised. Furthermore, Bio C8
1 to -30 (0,2 (z x 32cm) (Bio R
The reaction solution was passed through a solution (manufactured by AD) and eluted with 0 mM Tris-HCl (pH 7,3) A mM MgC1□ buffer to remove excess Kco Rnilinker.
Double-stranded cDNA with KcoRI linker was purified.

Using the obtained Kco RX linker-attached double-stranded CDNA fragment, 2Lt//DNA1 was cleaved with l2R1.
θμI and /Q double concentration of TIIDNAIJ gauze buffer (
(mentioned above) 10μt, 0. / MATP/ 0μt1T4D
10 u of NA ligase was added and the mixture was reacted for 4 hours in a reaction system with a total volume of 001Lt to insert the double-stranded cDNA fragment into 2Lt//DNA.

λ Phage Packaging Kit (Promega Biote
The above-mentioned DNA was introduced into the λ phage particles using the following method (manufactured by Ch. Co., Ltd.). The packaging procedure was performed according to the kit instructions.

This DNA panoke, λgt that has finished zinc / /
Using phages using conventional methods (Bacteriophage Experimental Methods 99 - /
The cells were infected with Escherichia coli Y1090 strain to form plaques according to the method of Keihin et al. Clone 1 was found to be positive by the immunoscreening method shown below from approximately 1 million plaques.
I got a piece.

The antibodies used in this immune screening were published by JP-A-Sho/,/
-/76g! It was prepared by the method described in Publication No. 1.

First, Y/090 infected with λgt//
gR, A, et al; Pro, Natl, Acad,
Sci, U,aA, (Grosseeding National Academy of Op Science U,S,A,),
go, / / q goo //9g, (/9g,?
)) was spread on a petri dish together with the upper layer of soft agar kept at 10°C, and left at 110°C in the evening. Then 10mV IP
A nitrocellulose filter containing TG (BA-g3 pore size o, 2 μm, manufactured by S&S) was placed thereon and cultured at 37° C. for 3 hours.

Cononitrocellulose fill l'-tTB8 buffer (
/ OmM Tris-HCI (1)H
7., t) Wash lightly with somM NaCl) and soak in TBS buffer poo- containing 3% gelatin.
The nitrocellulose filter was blocked by shaking at 10° C./hour. Next, a monoclonal antibody against non-A, non-B hepatitis specific antigen (0D28o=4
．． ,? ) in TBS buffer containing 7% gelatin.
Add it so that it is diluted by 7 parts, and put it in a plastic bag with the filters so that the dilution is 2 parts per 7 filters.
The reaction was carried out at room temperature for /A hours. Then O,OS%Tween
TBS buffer containing lIo 2.0.

Wash for 3 times for 10 minutes with a gelatin, and add a labeled anti-mouse xga-phP (7 oselate peroxidase) (manufactured by BiORad) to a TB containing 7% gelatin.
Add 100% dilution to S buffer, place in a plastic bag together with the filters so that 7 filters are diluted, and react at room temperature for several hours.
Washed three times for 10 minutes with TBS buffer ttoomt containing en 20. For color development, use a filter.
-/-naphthol (Bio Rac1 company split)
The sample was immersed in a 20-ml TBS buffer containing hydrogen peroxide.

After color development, the filter was thoroughly washed with water and stored in a cool, dark place in a plastic bag filled with water.

In this way, positive plaques were obtained. Single plaque isolation of this positive plaque was performed three times. Immune screening was performed in the same way all three times and it was confirmed that the results were positive.

Next, this phage was cultured in large quantities, and its DNA was purified. First, Y1090 bacteria were grown in NZ medium (NZ amine 109
, NaC1 evening El, ! r mMMgC1□ in water/l
(adjusted to pH 7.2)/Oml overnight. Infect this /rnt with phage so that it becomes m, o, i, (Multiple City Op Infection) O9/, leave it for IO minutes at 37°C, and then transfer it to NZ medium/1.
Culture with shaking at 37°C for 7 to 7 hours until the bacteria are lysed, then transfer to chloroform! - was added and shaking was continued for an additional 30 minutes. Next, remove the bacterial cell residue from step 4. ! ;00r,p,r
n, removed by centrifugation for 70 minutes and added NaCl to the supernatant.
After adding 29 g of polyethylene glycol and 70 g of polyethylene glycol and thoroughly dissolving the mixture, the mixture was left at 9° C. overnight. Otsu,! ;00 r,p,
Collect the precipitate by centrifugation for 20 min, thoroughly drain the water droplets, and transfer the precipitate to 20-7M buffer (10mM Tris-HC!1
(pH 7, &), 5 mM MgCl□), DNage and RNasθA were added to a concentration of 10μ, and the mixture was reacted at 37°C for 7 hours.

Next, 20-chloroform was added and stirred, and the polyethylene glycol was dissolved in the chloroform and removed from the aqueous layer. This aqueous layer was further heated for 2 r, 000 r, p
, m, ultracentrifugation for 0 min to obtain a pellet of phage particles. This pellet was dissolved in 7 ml of 7M buffer, and CsC1 density gradient centrifugation (33,00 Or, p,
m, , 20 hours) to obtain a fraction containing phage particles with ρ=/, q5~/, SO. After overnight dialysis against 7M buffer, proteinase K was purified by /θOμI/−
The mixture was added so as to give a reaction temperature of 100.degree. C. and reacted at 37.degree. C. for 7 hours. After that, the same amount of water-saturated phenol was added to perform gentle phenol extraction (after centrifugation for 5j0 Or, p, m, / 0 minutes, the aqueous layer was taken out, put into a dialysis tube, and dipped against water. Dialysis was carried out overnight at °C. In this way, approximately smg
DNA was obtained.

Transfer this DNA/00 μll to Kco R Engineering 10
When cleaved by reacting at 37°C with 100μ of the above-mentioned buffer system in 0u, the cD of J90bp and 3hiroSbp was detected.
It was found that the NA segment was inserted into the phage DNA. These λ Eco R fragments were cloned into the cloning vector pUC//9 Eco R
The commercially available primer CAGGAAAOAGOTATGAC! and AGTOACGACGTTGTA were used to determine the nucleotide sequence of each. The nucleotide sequence of the joining part of the two DNAs was determined by cutting the BamH engineering and Eco RV sites inside this cDNA fragment with restriction enzymes specific to the same sites, and the resulting BamHI-Sini RV DNA fragment was called pUo. The nucleotide sequence of the fragment was determined using the dideoxy method in the same manner by inserting the fragment into the BamHI site of //9.

The cDNA fragment has the base sequence shown in Figure 3. This was a partial cDNA fragment of a gene encoding a non-A, non-B hepatitis-specific antigen protein.Reference Example: Obtaining cDNA with a full-length geneReference Example/MRNA was prepared as described, and Okayama Depending on the vector, conventional methods (Molecular clonin
g.

cDNA is synthesized according to pp 2//, /qg, b).

The cDNA synthesis method is described below.

pCDV/(Okayama & Berg, Mob, eel, B
iO'l, (Molecular and Cellular Biology), Dan, 2go, (/qtr3))lI 0
0 ttFI / OmM Tris -
HCl (pH7,j), b m M M
A solution consisting of gC1□ and/OmM Naol, 70
0 ttL plus more! ;00 u of Kpn engineering (manufactured by Zenshuzo Co., Ltd.; all restriction enzymes are manufactured by Zenshuzo Co., Ltd. unless otherwise specified below) was added, and the mixture was reacted at 37° C. for 6 hours to cleave at the Kpn engineering site in the plasmid.

After phenol-chloroform extraction, DNA was recovered by ethanol precipitation. Approximately 200 μg of the DNA cut using Kpn was added to 10 mM sodium cacodylate.
Mu, ,? OmM Tris
- Hcl (IIIHt,,,g),
A buffer solution (hereinafter referred to as TdT) consisting of 7mM CaC1° and 0.1mM dithiothreitol (hereinafter abbreviated as DTT)
200 μt of a solution containing 0.23 mM dTTP (abbreviated as “buffer solution”), and further added f/u terminal deoxynucleotidyl transferase (hereinafter abbreviated as “TdT”) (PL Biochemical
a'ls Inc.) and reacted at 37°C for // minutes. Here, about 67 poly(dT) chains at the 3' end of the Kpn I cleavage site of pCDV/ were added. The solution was extracted with phenol-chloroform and precipitated with ethanol (
dT) strand-added pcDV/DNA approx./00111
1 was collected. The DNA'A was injected into io mM Tris-H.
CI (pH 7, &), 6mM MgC12 and / 00
In addition to the buffer solution/jOμt consisting of mM NaCl, 3 AOU ±1 μt was added, and the mixture was reacted at 37°C for λ hours. The reaction product was subjected to agarose gel electrophoresis, approximately 3,/K
Separate and collect the DNA fragment of p. Ottl's poly(dT) chain-added pcDV/ was obtained. 10 of the DNA
mM Tris-aal (1:lHg, θ) and /mMK
Dissolved in a solution SOOμt consisting of DTA, incubated for 65°C s, cooled on ice, and diluted with 50μt! r M N
a1l was added. The mixture was subjected to oligo(dA) cellulose column chromatography (manufactured by Collaborative Research). Poly(dT) with sufficient chain length is adsorbed onto the column, and this is transferred to /OmM) lis-MCI (p
It was eluted with a solution consisting of Hg, 0 ) and /mMKDTA to obtain 27 μg of pODV / (hereinafter abbreviated as vector primer) to which a poly(dT) chain was added.

Next, linker DNA was prepared. pL/(Oka
yama & Berg (Okayama & Berg) Mo1. Ce1L BioL (Molecular and Cellular Biology), 3.2KO, Cl9
g3)) approx./9μ77/OmM) Lis-H(
, l (pH 7,!r ), 6mM MgC1□ and!
A solution consisting of r OmM Mail + 200 μL was added, and SOU was further added thereto, reacted at 37° C. for 1 hour, and cleaved at the single step site in pL/DNA. The reaction product was extracted with 7 enols and chloroform, and then precipitated with ethanol, and about 3 μy of pL/DNA cut with PST was recovered. The final concentration of the DNA is approximately 0.2 μ/3μ #tl-TdT buffer! ; Solution 5θμ containing mM dGTP
In addition to t, T d T (PL Bioche
Yog unit (manufactured by Micals) was added and incubated at 37°C for 13 minutes to remove the Pst I cleavage site 3' of pL/.
Approximately 1/2 (aG) chain was added to the end. After phenol-chloroform extraction, DNA was recovered by ethanol precipitation. The DNA was dissolved in Lys-HCI (pH 7,5)
), A mM MgC1□ and A OmM NaCl
- in addition to 100 μt of buffer consisting of
Add lnd ll and incubate at 37°C for 3 hours.
The pL/DNA was cut at the Danjima ■ site.

The reaction product was fractionated by agarose gel electrophoresis.
A 3 Kb DNA fragment was extracted using the DEAF3 paper method (:
Dretzen et am (Dretzen et al.),
Anal.

Biochem, (analytical biochemistry).

//2,29 ri, (79 g/')) and linker DNA with oligo (dG) chains (hereinafter simply linker-D
(abbreviated as NA) was obtained. + (A) Approximately 2 μg of RNA per vector primer, approximately 50 mM Tris-HoI
(pHg,,y), g mM MgCl□, 30
mM KCI, 0.3mM DTT, 2mM dNTP (
dATP, dTTP.

dGTP and dCTP) and 10u of ribonuclease inhibitor (PL Biochemicals
10 u of reverse transcriptase (Seikagaku Corporation) was added and incubated at 37° C. for 90 minutes to synthesize NA complementary to mRNA. The DNA was extracted with phenol-chloroform and precipitated with ethanol to recover vector-primer DNA with an RNA-DNA double strand added thereto. 6 of the DNA
T containing 0μm dOTP and θ9.2μg poly(A)
Dissolve in 20μl of dT buffer and add /4u of TdT (P-L
Biochemica 1) was added and incubated at 37°C for 3 hours to incubate the cDNA with 72 (a
c) Added strands. The reaction product was extracted with phenol-chloroform, and the cDNA-vector-primer DNA with the (aC) chain added was recovered by ethanol precipitation.

The DNA was diluted with 10mM) Lis-HCl (pH 7,!; )
, A was dissolved in 1100 μt of a solution consisting of 1 mM MgC1
It was cut at one site. The reaction product was extracted with phenol-chloroform and precipitated with ethanol to obtain 0.5 pm of (a
c) Stranded cDNA-vector ply-r-DNA was obtained. The DNA O, Or pmole and the linker-D N A O,/A prnole were mixed at 10 mM.
Tris-HoI (pHq, s), 0. / M Na
Dissolve in q o ttt consisting of 1l and 1mM KDTA and incubate for 70 minutes at 65°C, Hiroko°C, and 0°C for 25 minutes.
and incubated for 30 minutes. 20 mM Tris-HoI (pH 7J), MMgC1□/Om
M(NH4)2So4.0,/MK(J and 0
．． A reaction solution was prepared so that the total amount was qooμt with a composition of /mM β-NAD. 10 u of E. coli DN was added to the reaction solution.
A ligase (manufactured by New England Biolabs) was added and incubated overnight. The reaction solution was treated with each IIoμM of dNTP, 0. /jmMβ-NA
Add the same ingredients to obtain D and add 1. tu E. coli D
N A IJ gauze, 7 u of E. coli DNA polymerase I (manufactured by P-LBiochemicals) and 2 u
Escherichia coli ribonuclease H (P-L Bioche
(manufactured by Micals) and incubate at /2°C and then at 5'C.
Incubated for hours.

In the above reaction, circularization of recombinant DNA including cDNA and RN
The RNA portion of the A-DNA duplex was replaced with DNA, producing a complete double-stranded DNA recombinant plasmid.

Escherichia coli MOIO prepared by a conventional method using this material
Competent cells of strain AII were transformed. Approximately 50,000 transformants were immobilized on nitrocellulose. These colonies were collected using the colony hybridization method [:Mo
1ecular CloningCold Spring
g harbor 1 laboratory PP,?
29 (79g)], the cDNA fragment obtained in Reference Example/ was labeled with 32p according to a conventional method and used as a probe. As a result of screening, three positive clones that strongly associated with each other at Dako°C were obtained.

As a result of detailed analysis of these clones by Southern's method (J, Mo1. Biol, (Journal Opmolecular Biology), yg, sθ3. Full-length c of the gene encoding the hepatitis-specific antigen protein
DNA was obtained. The expression vector containing this full-length cDNA is pC! DVC! It was named L-).

(Example/) Creation of A1 expression vector and transformant/) Mutation of N-terminus ■ pcDVCL-Ij td was mixed with 10 mM Tris-H
Cl (pa7.r), /θOmM NaO'l and Am
Digest by reacting with Pvul/Ou at 37°C for 2 hours in a buffer solution consisting of MMgC1□ (hereinafter abbreviated as "buffer H") tooμ, and heat at 7&0 for 1S minutes to inactivate the enzyme. After that, it was dialyzed against water and dried.

This was supplemented with J, 7 mM) lis-acetic acid (pH 7,
9), l-6m1j potassium acetate, / 0mM magnesium acetate and 0. ! ; 2mM'l-deoxytriphosphate was added to a buffer solution consisting of mM dithiothreitol;
The 3' protruding end was blunted using TF DNA polymerase lIu, the enzyme was inactivated by heating at 0°C for 10 minutes, and the enzyme was dialyzed against water. After drying, It was stored as a 0 μt aqueous solution.・・・・・・ Fragment I ■ Meanwhile, pcDV (L-120μs) was added to buffer
μ in Nco l, Hlnd, lll each 20
Digestion was performed at 37°C for 2 hours. this thing 5
% acrylamide gel electrophoresis (r9mM Tris,
g Buffer consisting of 9 mM boric acid and 2 mM EDTA 10 V/crn, /,! After separating the DNA using a 0.05% ethidium bromide aqueous solution, the fragments with larger molecular weights were cut out under 3'1 Onm ultraviolet light, and the gel was stained with a glass rod. After the disruption, the DNA was removed from the gel by suspending it in DNA extraction buffer (O,SM ammonium acetate, 10mM magnesium acetate, 0.7% lauryl sulfate, and 0.7% lauryl sulfate) and incubating it at 37°C/overnight. was extracted.

A large gel piece is 10,00 Or, p, m, /! After removing the gel by centrifugation for r minutes, remove smaller gel pieces using a glass filter, perform ethanol precipitation three times, and remove the DNA.
was purified and stored as a 200 μt aqueous solution. ...
・・ Fragment ■ ■ Use the primer for the part below that you want to mutate,
Synthesizer (manufactured by Nikkaki Co., Ltd., AI) 1)] -1edBiosy
It was synthesized using stem MODEL 3g0A).

The synthesized DNA was reacted with concentrated aqueous ammonia overnight at SS°C to remove the protective group, and then purified by reverse phase HPLO before use.

× × × [Original array E C---A--A-G
--TGG CAGTTA CAA CAAGAT--
------G-----TO-C-TAA CATGG
TTGCATG, -a ---L -------------)S/Base (x: displaced part) 50 pmol of the above primer was added to the kinase buffer (
! ; OmM) Lis-HCl (pHg, 0).

S' was phosphorylated with 20 u of T4 polynucleotide kinase in a 10 μt system (OmM MgCl□ and 5 nnM dithiothreitol).

■ Heteroduplex formation fragment IO, 0! r pmole, fragment] lo, ori pmole and S'-phosphorylated primer 4! ; Add polymerase ligase buffer (OJMNaOl, J 2.!) to the prnole at a concentration of S.
mM Tris-HOI (pH 7,!;)
, I/ OmM MgC1□ and jm
Add Mβ-mercaptoethanol) wo/2 μ, total, ?
'1. gμt system, boiled at 700°C for 3 minutes, immediately placed in a constant temperature bath at 30°C, and left for 30 minutes. next,
The mixture was left at 4° C. for 30 minutes and further left on ice for 70 minutes to form a heteroduplex.

Aqueous solution containing this heteroduplex //・tμt
Ku, jmM #-deoxynucleotide triphosphate was added to μt, /omMATP was added to 2/It, and 2u of KlenOW enzyme, 0,! ru T4 DNA ligase was added, and the reaction was carried out at 4° C. overnight in a total of 20 μt to circularize the DNA.

Using the aqueous solution λμt containing this circular DNA, Escherichia coli strain HB101 was transformed according to a conventional method to obtain a transformant.

A plasmid was isolated and purified from this transformant according to a conventional method, cut with restriction enzyme H1ndm, and separated into two fragments by 5% acrylamide gel electrophoresis to obtain a plasmid as a mutant plasmid. Since the mutant plasmid obtained in this case is often contaminated with the original plasmid (wild type), this mutant plasmid was used to transform Escherichia coli HB10/ strain again to purify the mutant plasmid.

In this way, plasmid pOV4+H was obtained (Figure 5
).

2) Modification of the C-terminus ■ 3 μg of pCDVCL-■ was treated in the same manner as in /)-■ to obtain 7 ragmen) I.
Fragment (2) was obtained in the same manner as (2) in /) above except that each sil &u was used.

■ Synthesize the following primer in the same manner as in (1) above, and use its S' phosphorylated 0glymer GOAOA.
AGGAAAAAATG [Original array] (---1--
-------------Axxxxxxxxxxxxxx GATATGTGAA A ------ACGTA
AATTTCC: lI B pace -----------
--1) (x: mutated part, *: added part) ■ Formation of heteroduplex In (1) of above /), fragments obtained in (2) to (2) above 1). Plasmid pcV'ltIB was obtained in the same manner except that n and y-phosphorylated primers were used (
Figure 6).

3) Introduction of specific antigen cDNA into expression vector ■ pO
VQ4H/θ μg (~3 pmol) was added to buffer H/'
o o μ inside, HindllJθu z shishi 1.
Using 20 u, the mixture was reacted at 37° C. for 2 hours and then cut.

This was subjected to 5% acrylamide gel electrophoresis, and Q
A 7 bp DNA encoding the N-terminus of a specific antigen
The fragments were isolated and purified. ...Fragment N ■ pOVV<zB t ottgc ~,? pm
ol) in buffer solution H/θoμ.
Using 20U of ACL, react at 37°C for 2 hours,
Amputated. This material was subjected to 5% acrylamide gel electrophoresis, g, ? A DNA fragment encoding the C-terminus of the Abp specific antigen was isolated and purified. ...Fragment C ■ Expression vector pUs△H2μg (~/pmo
l) in buffer H and ind l 2u , Bq1
Using 112 u, reaction was carried out at 37° C. in a microt system and cleavage was performed. This product was extracted with the same volume of water-saturated phenol to remove protein, and the phenol was extracted with ether, followed by dialysis against water, desalting, and concentration using a vacuum pump to obtain the expression vector fragment HB. lOμt containing
An aqueous solution of was obtained.

■ Fragment NO,! r pmol, fragmen) CO,! ; pmol and expression vector fragment HB
O, /pmo1, /OmM Tris-Ho
I (pH 74), /mM dithiothreitol, A m
M 10μ of buffer consisting of Mg0I□ and /mMATP
T-Hiro DNA ligase/U was added to the mixture, and the mixture was reacted at 9°C for 6 hours. Using 3μ of this product, commercially available E. coli JM109 competent cells were transformed according to a conventional method. Transformants were selected in L medium (Bactobeptone 709/L, Yeast Extract 51!/L, NaC1/01//L, Agar tsg/1.) containing 20 μEl/ml of acipicillin, and the cells containing the specific antigen gene were selected. Expression vector pOZ14! was obtained (Figure 7).

B, Expression of specific antigen E. coli 1M109 strain harboring pcZ'l'l
The cells were cultured overnight at 30°C in a gloss. This material was inoculated into a new L-gross medium at a 5θ-fold dilution, and incubated for 2 hours.
Culture was performed with shaking at 30°C. Next, PTG (isopropyl-β-D-galactopyranoside) was added to the medium to a concentration of 2mM, and cultured with shaking at 30°C for an additional 3 hours. Bacteria were collected by centrifugation at r 00 r, p, m for 10 minutes. This is 0.9% Ma
Confirmation of the expression of the OC8-specific antigen. ．． 3 ml culture, 10%
SDS polyacrylamide gel electrophoresis (Tris, 3!
/L, glycine/Da, GEl/, and a buffer consisting of 0, /% SDS, /20, for 7 hours), then the gel was taken out and layered on a nitrocellulose filter, sandwiched between filter paper, and filtered with Tris. 311/l, glycine/Hiroshi, 9I/
The gel was run at 5 V/crn and 1° C. in a buffer solution of 5 V/l, and the protein in the gel was plotted on a nitrocellulose filter. The nitrocellulose filter was soaked in TBS buffer (10mM Tris-HOI (pHq,
s) and left OmM Mail) and TBS buffer containing 3% gelatin.

The nitrocellulose filter was immersed in - and shaken for an hour at θ°C to remove the nitrocellulose filter. Next non-A non-B
Monoclonal antibody against hepatitis-specific antigen (OD28
o = g, 3) in TBS buffer containing 7% gelatin.
The mixture was added to a dilution of 0.00 min/diluted, placed in a plastic bag together with the filters at a ratio of 2 to 7 filters, and allowed to react at room temperature for 76 hours. Next 0.05% Twee
Washed 3 times for 70 minutes with TBS buffer containing ncoO, then labeled secondary antibody anti-mouse egg, a-PAP.
(Force Radish Peroxidase) (Bio
(manufactured by Rad) Add it to TBS buffer containing 7% gelatin at a 100-fold dilution, place it in a plastic bag together with the filters so that the ratio is 2-2 for every 7 filters, react at room temperature for several hours, and in the same way with O ,OS%Twee
TBS buffer containing 20 lIo.

- for 10 minutes three times. Use a filter for color development
-chloro-/-naphthol (Bi.

(manufactured by Rad) / 20-TB containing hydrogen peroxide solution
It was immersed in S buffer. After color development, the filter was thoroughly washed with water and stored in a cool, dark place in a plastic bag filled with water.

In this way, we investigated the presence or absence of the expression of non-A, non-B hepatitis-specific antigens, and a protein that reacts with the same position (lI+Xa) as the specific antigen derived from infected chimpanzee liver was detected.
Expression of the specific antigen was confirmed in E. coli.

(Effect of the invention) When using non-A, non-B hepatitis-specific antigen protein directly as a diagnostic reagent, a large amount of non-A, non-B hepatitis-specific antigen protein is required; It is difficult to purify specific antigen proteins from hepatocytes of chimpanzees and the like at the onset of non-A, non-B hepatitis. According to the present invention, a large amount of the antigenic protein can be purified safely and inexpensively without using infected hepatocytes of chimpanzees or the like.

[Brief explanation of the drawing]

Figure - / is a diagram showing the base sequence encoding a non-A, non-B hepatitis-specific antigen protein. In the figure, "-" represents a base complementary to the base shown above. Figure-λ is a diagram showing the base sequence of the hybrid promoter Pac. Figure 3 is a diagram showing the base sequence of cDNA obtained in Reference Example/. Figure 1 is a diagram showing the base sequence of cDNA obtained in Reference Example Y. In the figure, numbers 57 to 1.3gg represent the base portion encoding the non-A, non-B hepatitis-specific antigen protein. Figure 5 shows plasmid pCV! Represents a schematic diagram for creating 4H. Figure 6 represents a schematic diagram of the construction of plasmid pOV94B. Figure-7 represents a schematic diagram for creating plasmid pCZ 1111. Applicant: Mitsubishi Chemical Industries, Ltd. Agent: Patent Attorney Nagakagawa - 7 others Law 1 +n 1QA (Zone 1) ■ 5-1 (Zone 2) GAA AAT CTT GGAAA GGG
GTCATT AnC:- Knee I CTG CTG GGT CCA ATT('
e/)3') TAT AGG ACA TACGAT GG
CAAA TAC GACTCA CTG GGG TCT ATT AGA GACGGG AA
ACTG CCA TTT ATT CTG
TGTCTG AGT GAG AAA GA
A GGC-1 (So/74) Note-1 1 ('f/)5) ff1-3 Sakura no 1) CTA GGA CTA TAT ACA CCA
GAA ACA CTG TTTLeu Gly L
eu Tyr Thr Pro Glu Thr
Leu Phe +Thr Asn Phe Gln
Ile Asp Gly Arg Asn Arg
1AAT CTT GGA CTT GCT CAA
AAT TGT ACT ATC':l:ys Cy
s Asp Vat Ala Lys Tyr As
n Ser Pr. ToAA GTG ATT ATG GACTT
A AAG ACA ATG GAA=ys
Val IIs Met Asp Leu Ly
s Thr Mat GluTCT ATT CAG
GAT TAT GAA GTT TTT CGA
TGCMan L! llu La1y Llu
/410 (jln ASn Cys I
nrGAA GAT TCA CTG GACGAA
AGA AAG ATA. Glu Asp Ser Leu Asp Glu A
rg Lys Ile 1Ser Ala Leu
Arg Thr Tyr Glu Pro Tyr (
CTG GGT CCA ATT GGA GCT
GGG AAG TCT rLeu Gly Pro
Ile Gly AIOGly Lys Sir!
GGG CAT GTAACG CAT CAG GC
T TTG GTG (Gly His Val T
hr His Gln Ala Leu Val (A
Ie Ser Ile Gln Asp TY
r Glu VOI Pr1lil Arg
cysAAA GGG GTCATT GAG CT
CAGG AAG AGCTTA CTGLys Gl
y Vat Ile Glu Leu Arg L
ys Ser Leu Leu31y Ser Leu
Val Gln Gln IIs Arg Ile
Leu Leu to GCTTT TTCAACTCA
GTG AGG TCT GTT TTCCA
A3・r Phe Pha Asn Ser Vat
Arg Sir Val Phi G1n3GCAC
T AAT ACA ACT GGG ATA TCT
GAG AAG TAT31y Thr Asn T
hr Thr Gly Ile S+! r Glu L
ys Tyr-3 (Uke no 2) 670 田0690 AGG ACA TACTCT ATT AGA G
ACGGG AAA GAT IArg Thr Ty
r Ser IIs Arg Asp Gly L
ys Asp 1TCA CTG GGG CT
G AGT GAG AAA GAA GGCG
GCl5er Leu Gly Leu Sir
Glu Lys Glu Gly Gly 1A
ACGGT AACATT CGT GAT AG
A TACCAG TTT. Asn Gly Asn xle A "g Alp
A'g Tyr Gln Phe 1CAT GACT
ACATT GAT TCCCCA TCG CTG
AAG700 7+0
7203GCAAA TACCTG CCA TT
T ATT CTG TGT GACGly Lys
Tyr Lau Pro Phi Ile Leu
Cys Asp:TG TGCATG GAT G
ACATA TCCTACATCTTGLeu Cys
Met Asp Asp Its Ser Tyr
Its Leu8AT CCCATG GAA
TCA ATCAAA TTA AAT CA
T to sn Pro Met Qlu Ser
lle Lys Leu Asn HisGA
CAGA ATT CAT TGT GTG GCA
TTT GTA TTTH His Asp Tyr
Ile Asp Ser Pro! 3er Leu
LysGAT GCCAGCTCT ATT GAA
TACTTCTCCTCTAsp Ala Ser S
er IIs Glu Tyr Phi Ser
5erAGG GAG TTG GTA AACGC
T GGT GTG GTA CATArg Glu
Leu Val Asn Ala Gly Val
Vat His1030 1040
+050GAT CTG ATT AC
A AAA GGT GACCTT ATA GAAA
SpLllu 1+e 7hr LJs Gly AL
P lju 工＋e QluAsp Arg Tle
His Cys Val Ala Phe Vat
PheCAG ATG ATA GTA AAG
ATCAAA AGA ATT CGAGin Me
t Ile Val Lys lle Lys
Arg Ile Arg+000 1
010 +020GTG GCT
TTG CTCACT CAT GTG GAT A
GCATG Vat Ala Leu Leu Th
r His Val Asp Ser MetAT
AGAG AGA TGT GTG CCT GTG
AGG TCCAAGIll! Qlu ArQ CI
VOI ProVal Arg Ser Lys knee 3 (Nobu no 3) 1090 1+00 1
110CTA GAG GAA GTCCAA
AGA AAA CTT GGA TTTLeu
Glu Glu Val Gin Arg
Lys Leu Gly Phe. +150 +160
1170TAT TCCTCT GAG TGG
GAG CTG GACCCT GTA1120
1130 +140GC
T CTT TCT GACATCTCG GTG
GTr AGCAATala Leu Ser
Asp Ile Ser Val Val Ser
AsnAAG GAT GTT CTA ATT
CTT TCT GCT CTG AGAlyr 5
er “5er 1.ilu lrp (jl
u Leu Asp Biro VOI Ly
CGA ATG CTA TGG GCT G
CA GAT GACTTCTTA GAArg Me
t Leu Trp Ala Ala Asp A
sp Phe Leu G11270
1280 +290CTA AGG
GAG GAA ATT ATCAACTGT GCA
CAA GGLeu Arg Glu Glu Il
e Engineering 1e Asn Cys Ala Gln GlS
Asp V (ml Leu 114! Le
u S4! r AIG Leu Ar9+240
1250 1260, G
GAT TTG CCT TTT GAG CAA
ATA GGG AATu Asp Leu Pro
Phe Glu Gin Ile Gly Asn
iA AAA AAA TAG y LysLys IX! ! B-4 (1 of A) +0 20 30
405' GGGGGGCTACCCTC
AGCTCT AGCTCATACT ACAGACA
GTA3' CCCCCCGATG GGAGTCG
AGA TCGAGTATGA TGTCTGTCA
T90 too 110
+20TGGTTGCATG AA
AAGATCCT GCAAAATCAT TTTG
GAGGGA. ACCAACGTACTTTTCTAGGA CGT
TTTAGTA AAACCTCCCCT+70
180 +90
200CCATAATGGA GTTTTGCTTG
ACAGATGTTG TAATCAAGGGGGT
ATTACCT CAAAACGAACTGTCTA
CAACATTAGTTCCCGAGCATATGCA
GAAGAGGGT TACCAGGAAA GAAA
GTATGCCTCGTATACGTCTTCTCC
CA ATGGTCCTTT CTTTCATAC
GCAACAGATCAAGAAGTATGG C
AGTGACAACTCGTTTGACAGTTGTC
TAGT TCTTCATACCGTCACTGTTG
to AGCAAACTGTGCGGCTTAG C
CTTCTCTAT AAGGGTAGTG TC
CATGGATTTCGCCGAATCGGAAGAG
ATA TTCCCATCACAGGTACCTAA
2I0 220 230
240290 300
3+0 320TTCCATCATCCT
TTTTGCACTTCAAAGAGACTAAAATT
TCAAAGGTAGTAG GAAAAAACGTG
AAAGTTCTCTG ATTT Engineering AAAGTGAA
TGGAAAACTAGGACTATA TACACC
AGAAACACTGTTTTCTTACCTTTT
G ATCCTGATAT ATGTGGTCTT
TGTGACAAAAGATAGATGGA AGA
AATAGAA AAGTGATTAT GGAC
TTAAAGCTATCTACCTTCTTTAT
CTT TTCACTAATA CCTGAATT
TCCTATTCAGGA TTATGAAGTT
TTTCGATGCG AAGATTCACTGA
TAAGTCCT AATACTTTCAA AAA
GCTACGCTTCTAAGTGAAGCTTACT
GT CTGCCTTGAG AACTTATGA
A CCATATGGATTCGAATGACA
GACGGAACTCTTGGAATACTT GGT
ATACCTAGTTGTGACGTTGCAAAA
TAT AACTCCCCAA CTAATTTCC
ACAACACTGCAACGTTTATATT
GAGGGGTT GATTAAAGGTACAAT
GGAAAATCTTTGGACTTGCTCAA
AAT TGTACTATCTTGTTACCT
TAGAACCTGA ACGAGTTTTA
ACATGATAGAGGACGAAAAGAAAG
ATAAAAG GGGTCATTGA GCTC
AGGAAGCCTGCTTTCTTTCTATT
TTCCCCAGTAACT CGAGTCCTTC
CCCTGGTTCA ACAAATACGA AT
TCTGCTGCTGGGTCCAATGGGACCA
AGT TGTTTATGCT TAAGACGA,
CG ACCCAGGTTA! -4 ((Q2) TGGAGCTGGG AAGTCTAGCT T
TTTCAACTCAGTGAGGTCT 1ACCT
CGACCCTTCAGATCGAAAAAGTTG
AG TCACTCCAGACTAATACAACT
GGGATATCT GAGAAGTATA GG
ACATACTC'GATTATGTTG ACCCT
ATAGA CTCTTCAT'AT CCTGTAT
GAGCTGTGTGACT CACTGGGGCT
GAGTGAGAAA GAAGGCGGCCG
ACACACTGA GTGACCCCGA CT
CACTCTTTCTTCCGCCGGTCGTG
ATAGA TACCAGTTTA ATCCCA
TGGA ATCAATCAAA'AGCACTA
TCT ATGGTCAAAT TAGGGTACC
TTAGTTAGTTT rACAGAATTCA
TTGTGTGGCA TTTGTATTTG A
TGCCAGCTC'TGTCTTAAGT AACA
CACCGT AAACATAAAACTACGGTC
GAGGTTTTCCAAG GGCATGTAAC
GCATCAGGCT TTGGTGGGCACAAA
AGGTTCCCGTACATTG CGTAGTC
CGA AACCACCCGTTATTAGAGACG
GGAAAGATG GCAAATACCT GCC
ATTTATTATAATCTCTG CCCTTTC
TACCGTTTATGGA CGGTAAAATAA
TGTGCATGGA TGACATATCCTACA
TCT Engineering GA ACGGTAACATACACGTAC
CT ACT GTA Ding AGG ATGTAG
T TGCCATTGTArTAAATCATCAT
GACTACAT TGATTCCCCA TCGC
ATTTAGTAG TACTGAT to TGAAGG
GTA ACTAAGGGGT AGCGACTT
CClolo 1020 10
30 +040TATTGAATACTT
CTCCTCTCCAGATGATAGTAAAGAT
CAAAATAACTTATG AAGAGGAGAG
TCTACTATCATTTCTAGTTT+05
0 1060 1070
+080AGAATTCGAAGGGA
GTTGGT AAACGCTGGT GTGGTA
CATG ”TCTTAAGCTT CCCTCAA
CCA TTTGCGACCA CACCATGTA
Cr1130 1140 115
0 +160AAAAGGTGACCTT
ATAGAAA TAGAGAGATG TGTGC
CTGTGTTTTCCACTG GAATATCT
TT ATCTCTCTACACACGGACAC12
1012201230+240 CTCTTTCTGA CATCTCGGTG G
TTAGCAATT ATTCCTCTGAGAGAA
AGACT GTAGAGCCACCAATCGTT
AA TAAGGAGACT+090 1
100 1110 1+20rG
GCTTTGCT CACTCATGTG GATA
GCATGG ATCTGATTAC to CCGAAAC
GA GTGAGTACACCTATCGTACCT
AGACTAATGl 170 1180
1190 +200AGGTCC
AAGCTAGAGGAAGT CCAAAGAAAA
CTTGGATTTGTCCAGGTTCG AT
CTCCTTCA GGTTTCTTTT GAACC
TAAAC125012601270+280 GTGGGAGCTG GACCCTGTAA AG
GATGTTCT AATTCTTTTCTCACCCT
CGACCTGGGACATTTCCTACAAGA
TTAAGAAAGA-4 (A-3)

Claims (7)

[Claims] (1) An expression vector characterized by introducing a DNA fragment containing a DNA encoding a non-A, non-B hepatitis-specific antigen into a cloning site located downstream of a promoter. (2) The expression vector according to claim 1, wherein the promoter is controllable by a regulatory element. (3) The expression vector according to claim 1, wherein the promoter is a promoter that functions in microorganisms. (4) The expression vector according to claim 1, wherein the promoter is a promoter that functions in eukaryotes. (5) D that encodes a non-A, non-B hepatitis-specific antigen
1. A transformant obtained by transformation with an expression vector obtained by introducing a DNA fragment containing NA into a cloning site located downstream of a promoter. (6) The transformant according to claim 5, wherein the host is E. coli or Bacillus subtilis. (7) A DNA fragment containing a DNA encoding a non-A, non-B hepatitis-specific antigen is introduced into the cloning site downstream of the promoter of the expression vector, and then the D
A method for producing a non-A, non-B hepatitis-specific antigen, which comprises introducing an expression vector into a host into which an NA fragment has been introduced, culturing the host, producing and accumulating the antigen, and obtaining the antigen.