JPH06225770A - Nucleic acid fragment coding non-a non-b hepatitis virus antigen - Google Patents

Abstract

PURPOSE:To obtain a new nucleic acid fragment useful for the nucleic acid diagnosis of non-A non-B hepatitis patient and the production of a recombinant non-A non-B hepatitis virus antigen (poly)peptide. CONSTITUTION:The nucleic acid fragment contains a nucleotide sequence coding the structural and non-structural regions of non-A non-B hepatitis virus obtained from the blood plasma of a non-A non-B hepatitis patient by genetic engineering technique. It is expressed e.g. by a part or total of the amino acid sequence of formula. An RNA is extracted from the blood plasma of a non-A non-B hepatitis patient, cDNA is collected therefrom and amplified by PCR using two kinds of primers, the obtained DNA is cloned with a cloning vector replicative in E.coli and the nucleotide sequence is determined by Sanger's dideoxy-chain termination method.

Description

Detailed Description of the Invention

[0001]

FIELD OF THE INVENTION The present invention relates to nucleic acid fragments encoding antigens of non-A non-B hepatitis virus structural and non-structural regions. The present invention also relates to an expression vector containing the nucleic acid fragment and a host cell containing the vector. The invention further relates to a method for producing said antigenic (poly) peptide and the recombinant (poly) peptide obtained thereby.

[0002]

2. Description of the Related Art Generally, hepatitis A virus, which is orally infected, and hepatitis B virus, which is infected through blood, are widely known as viruses that cause viral hepatitis.
In addition, type D (δ), which is associated with hepatitis B virus
The presence of the hepatitis virus is also known.

In addition to these, there has been hepatitis whose infectivity has been pointed out for a long time but the existence of its causative factor was unknown, and the existence of multiple viruses was suggested.
It was collectively referred to as non-A non-B hepatitis due to the presence of the hepatitis B virus and the exclusion diagnosis of other hepatitis factors. Recently in this
A non-A non-B hepatitis virus that causes hepatitis by oral infection has been isolated and identified.

On the other hand, non-A which is transmitted mainly through blood transfusion
As for non-hepatitis B, hepatitis B virus development and
The prevention of blood transfusion for blood transfusions has made it possible to prevent most of them. At present, more than 90% of post-transfusion hepatitis, more than 50% of infected people become chronic, and the rate of transition to liver cirrhosis and liver cancer is high, which is a serious problem. It was.

[0005] Regarding this virus, in 1989, Choo et al. Of Chiron, USA cloned a virus gene by immunoscreening method using chimpanzee plasma infected with human plasma as a sample, and used a microorganism based on the cloned virus gene. Developed a diagnostic method by antibody test using expressed antigen (Science 244:
359-362 (1989); Science 244: 362-364 (1989); Japanese Patent Publication No. 2-500880). With this as an opportunity, active research has begun all over the world, and the entire primary structure of the virus has been clarified (Proc. Natl. Acad. Sci. 87: 9524 (199
0); Proc. Natl. Acad. Sci. 88: 2451 (1991); J. Viro
l. 65, 1105 (1991)), currently widely used by Hepatitis.
It is called by the name of C virus (HCV). However, the c10 originally developed by Chiron
With the reagent using 0-3 antigen, only 70 to 80% of chronic non-A non-B hepatitis patients could be detected (Shiro Iino et al., Medicine and Pharmacy 26 (1): 87-95, 1991). However, since then, the HCV gene has been actively cloned not only in the United States but also in Japan, and it is possible to detect almost 90% of patients by developing a second-generation reagent containing the core antigen that is a viral structural protein. (Kawai Tadashi et al., Clinical laboratory equipment / reagents 14 (4): 725-733, 1991). However, even with this improved second-generation reagent, only about 40% thereof is detected in sporadic non-A non-B hepatitis patients.

[0006] On the other hand, with the progress of research on HCV, it has been pointed out that there are some genes having very different homologies among viral genes, and it is being considered that they may be divided into at least two or more genotypes (Virus. Gene
5: 3, 243 (1991); Proc. Natl. Acad. Sci. 88: 10292 (1
991)).

[0007]

As described above, non-A
Although a significant portion of non-hepatitis B patients have been diagnosed by second generation, HCV antibody detection reagents combining structural and non-structural region antigens, there are still patients who cannot be detected by these reagents. This cause is
Is it due to different types of non-A non-B hepatitis virus,
It is not clear whether it is due to another pathogenic factor.

[0008] In addition, non-A non-B such as interferon administration
With the advent of therapeutic methods for hepatitis B patients, more meaningful gene and antigen measurements are strongly desired not only for detecting antibodies but also for determining therapeutic effects. However, it is becoming clear that there are different types of non-A non-B hepatitis viruses, and it is also becoming clear that there is considerable diversity, especially in the region of the envelope. When performing antibody measurement, antigen measurement, or gene measurement as an index of virus infection, it is considered necessary to consider the diversity of viral antigens and genes, and in order to do so, use as many types of viral genes as possible. It is considered necessary to obtain the expression product.

The object of the present invention is to provide novel nucleic acid fragments encoding the antigens of the non-A non-B hepatitis virus structural and non-structural regions.

Another object of the present invention is to provide an expression vector containing the nucleic acid fragment.

Still another object of the present invention is to provide a host cell containing the expression vector.

[0012] Another object of the present invention is to provide a method for producing the antigen (poly) peptide obtained by culturing the host cell and expressing the nucleic acid fragment.

[0013]

[Means for Solving the Problems] In order to achieve the above-mentioned object, the present inventors have selected a non-A non-B hepatitis virus gene different from the previously reported one from the plasma of a specific non-A non-B hepatitis patient. It succeeded in cloning, and came to complete the present invention.

In completing the present invention, non-A non-B
RNA is extracted from hepatitis C patient plasma, reverse transcriptase is acted to obtain cDNA, and PCR is performed using two kinds of primers.
(Polymerase chain reaction; Science 230: 1350 (1985)) to amplify the DNA. For the primers used for amplification, see the previously reported sequence (J. Virol. 65,
1105 (1991); Proc. Natl. Acad. Sci. 87: 9524 (1990);
Virus Gene 5: 3 243 (1991); J. General Virol, 72: 269.
7 (1991)). The amplified DNA was cloned using a cloning vector capable of replicating in E. coli, and the Sanger dideoxy chain termination method (Science,
214, 1205 (1981)) was used to determine the nucleotide sequence.

Five kinds of clones were obtained by the above method,
C6-62, C6-66, C6-79, C6-82, respectively
And C260-1. Incidentally, C6 and C260
Is a series of clones derived from HCVR6 strain and HCVR260 strain, each obtained from a single patient. The obtained 5 types of clones are respectively under the accession number FERM P-
13707, FERM P-13708, FERM P
-13709, FERMP-13710 and FERM
Deposited as P-13711 on June 28, 1993 at the Institute of Biotechnology, Institute of Biotechnology, AIST.

As shown in FIG. 1, the obtained 5 kinds of clones were homologous to the nucleotide sequence of the previously reported non-A non-B hepatitis virus gene, and C260-1 was the core / E1 region, and C6-62 was the same. , C6-66, C6-79 and C6-82 were presumed to be NS2-NS5 regions. Determined clones C6-62, C6-66, C6-79, C6
The amino acid sequences deduced to be the nucleotide sequences of -82 and C260-1 are shown in SEQ ID NO: 1 in the sequence listing below,
Shown in 2, 3, 4 and 5.

The characteristics of each of the obtained clones are shown below.

(1) Clone consisting of C6-62 1755 nucleotides, the translation region is nucleotide numbers 1 to 1755 (585 amino acids), and NS2
And part of the NS3 and NS4 regions.

(2) Clone consisting of C6-66 1451 nucleotides, the translation region is nucleotide numbers 2-1450 (483 amino acids), NS5
It corresponds to a part of the area.

(3) Clone consisting of C6-79 2093 nucleotides, the translation region of which is 3 to 209.
3 (697 amino acids), NS3 and NS4, N
It corresponds to a part of the S5 area.

(4) Clone consisting of C6-82 1386 nucleotides, the translation region is nucleotide numbers 1 to 1386 (462 amino acids), NS5
It corresponds to a part of the area.

(5) Clone This consists of C260-1937 nucleotides, and the translation region is nucleotide numbers 2 to 937 (312 amino acids), which corresponds to a part of the core and E1 regions.

The non-A non-B hepatitis virus genome coding region is composed of a core / envelope structural region and a non-structural region (NS). CORE-E1-E2 / NS1-NS2-NS3-N
They are arranged in the order of S4-NS5 (J. Virology (199
1), 65: 1105-1113).

Further clones C6-62, C6-66, C
The nucleotide sequences and deduced amino acid sequences of 6-79, C6-82, and C260-1 are reported in previously reported HCV1.
(Proc. Natl. Acad. Sci. (1991), 88: 2451 to 2455),
HCVBK (J. Virology (1991), 65: 1105 to 1113),
HCV-J1 (Proc. Natl. Acad. Sci. (1990), 87:95.
24-9528), HC-J6 (J. General Virology (199
1), 72: 2697-2704) and HC-J8 (Virology (199
2), 188: 331 to 341) and the results of comparison of homology are shown in Tables 1, 2, 3, 4 and 5 below.

[0025]

[Table 1] Table 1 Homology (%) HCV gene All nucleotide sequences All amino acid sequences C6-62 / HCV1 79.5 90.8 HCVBK 91.4 95.7 HCV-J1 70.1 81.3 HC-J670 .2 81.3 HC-J8 70.5 80.2

[0026]

[Table 2] Table 2 homology (%) HCV gene entire nucleotide sequence the entire amino acid sequence C6-66 / HCV1 74.7 80.0 HCVBK 91.9 95.2 HCV-J1 58.8 60.5 HC-J6 59 0.0 61.2 HC-J8 60.5 59.9

[0027]

Table 3 Table 3 Homology (%) HCV gene All nucleotide sequences All amino acid sequences C6-79 / HCV1 78.8 85.5 HCVBK 92.4 96.1 HCV-J1 66.8 73.3 HC-J6 66 .8 73.9 HC-J8 66.6 73.3

[0028]

[Table 4] Table 4 Homology (%) HCV gene All nucleotide sequences All amino acid sequences C6-82 / HCV1 82.5 87.1 HCVBK 93.2 94.4 HCV-J1 70.8 73.4 HC-J6 71 .0 74.5 HC-J8 70.2 73.1

[0029]

Table 5 Table 5 Homology (%) HCV gene All nucleotide sequences All amino acid sequences C260-1 / HCV1 71.9 75.0 HCVBK 70.8 73.4 HCV-J1 79.0 83.3 HC-J6 79 .0 83.3 HC-J8 92.3 95.2 From these tables, clone C6-62 shows the published non-A.
Nucleotide sequence of 8.6 to 29.9% and amino acid sequence of 4.3 to 40. with non-hepatitis B virus gene.
Clone C6-66 shows 8.1% to 41.2% and 4.8 to 40.1% respectively; clone C6-79 shows 7.6 to 33.4% and 3.9%, respectively. ~
26.7%; 6.8-for clone C6-82, respectively.
29.8%, 5.6-26.9%; and clone C26.
0-1 was 7.7 to 29.2% and 4.8 to 2 respectively.
A difference of 6.6% was observed. This means that HCVR6
Strain and HCVR260 strain have been published to date.
It indicates that the strain is different from the CV strain.

Accordingly, the present invention provides a novel nucleotide sequence encoding the structural and non-structural region antigens of non-A non-B hepatitis virus obtained by genetic engineering from plasma of a non-A non-B hepatitis patient. A nucleic acid fragment is provided.

According to a specific embodiment of the present invention, the nucleic acid fragment is a non-A non-B hepatitis virus represented by all or part of the amino acid sequences shown in SEQ ID NOs: 1, 2, 3, 4 and 5 in the sequence listing below. It includes a nucleotide sequence that encodes an antigen.
In addition, the nucleotide sequence includes all sequences based on the degeneracy of the genetic code. Specific examples of such a nucleotide sequence include all or part of the sequence of nucleotide numbers 1 to 1755 shown in SEQ ID NO: 1, all or part of the sequence of nucleotide numbers 2 to 1450 shown in SEQ ID NO: 2, All or part of the sequence of nucleotide numbers 3 to 2093 shown in SEQ ID NO: 3, all or part of the sequence of nucleotide numbers 1 to 1386 shown in SEQ ID NO: 4, from nucleotide number 2 shown in SEQ ID NO: 5 All or part of the sequence up to 937.

The present invention also provides an expression vector in which the above-mentioned nucleic acid sequence is introduced into the cloning site in the vector existing downstream of the promoter. Furthermore, the present invention provides a host cell containing the expression vector.

As the vector, a virus (eg, vaccinia virus, baculovirus, etc.) can be used in addition to a commonly used vector such as a plasmid or a phage. D
The type of promoter and host that can be used depends on whether or not the recombinant (poly) peptide obtained by NA expression has a sugar chain structure. That is, when the recombinant (poly) peptide does not contain a sugar chain structure, a prokaryote such as Escherichia coli, Bacillus subtilis, actinomycete can be used as a host, and tryptophan synthase can be used as a promoter. Operon (trp), lactose operon (lac), lambda phage P _L , P
_R etc. can be used. In this case, it will generally be obtained as a fusion with another peptide. On the other hand, when the recombinant (poly) peptide has a sugar chain structure, examples of the host include eukaryotes such as yeast, plant cells, insect cells, and animal cells, and a promoter commonly used in yeast and the like. Promoters such as promoters for glycolytic enzymes such as 3-phosphoglycerate kinase and enolase, promoters for alcohol dehydrogenase, viral promoters that can be used in mammalian cells such as polyoma virus, adenovirus, simian virus SV40, vaccinia virus, site Examples include promoters derived from megalovirus and the like. Normally,
Prokaryotes are used as hosts, and thus promoters suitable for prokaryotes are used.

The vector further comprises a marker sequence (for example, ampicillin, tetracycline resistance gene, etc.) which enables phenotypic selection of transformed cells, an origin of replication,
It may appropriately contain a terminator, a ribosome binding site and the like.

The present invention further provides a method for producing a recombinant non-A non-B hepatitis virus antigen (poly) peptide. Specifically, this method comprises the steps of constructing a replicable expression vector capable of expressing the above-described nucleic acid fragment of the present invention in a suitable host cell, introducing the expression vector into the host cell, and transforming the expression vector. The step of obtaining the above-mentioned recombinant (poly) by culturing the transformant under conditions capable of expressing the nucleic acid fragment.
The step of expressing the peptide, and the step of recovering the recombinant (poly) peptide.

The culture conditions of the transformant are determined depending on the host cell used, and a culture medium capable of growth, culture temperature, culture time and the like are appropriately selected. The recombinant (poly) peptide can be purified from the culture by conventional techniques such as ultrasonic disruption of cells, solubilization extraction, ammonium sulfate fractionation and various chromatographies.

The term "recombinant (poly) peptide" as used herein is obtained by expressing a DNA encoding a non-A non-B hepatitis virus antigen incorporated in an expression vector (poly).
By peptide itself or a fusion (poly) peptide with another (poly) peptide is meant.

The present invention also includes a recombinant (poly) peptide obtained by the above method. Such (poly) peptide can also be chemically synthesized by using a conventional peptide synthesis technique, which is obvious to those skilled in the art.

The recombinant polypeptide obtained by the present invention was subjected to, for example, SDS-polyacrylamide gel electrophoresis and then subjected to Western blotting to obtain normal human serum and non-A.
When reacted with non-hepatitis B patient serum, this recombinant polypeptide reacted only with non-A non-B hepatitis patient serum. Therefore, this recombinant polypeptide is an antigen specific to non-A non-B hepatitis virus and can be used for diagnosis of non-A non-B hepatitis and detection of non-A non-B hepatitis virus.

[0040]

The present invention will be described in more detail by the following examples, but the present invention is not limited to these examples.

Example 1 Detection of HCV (# R6) Gene by RT-PCR As a method for cloning the HCV gene from plasma of patients with chronic non-A non-B hepatitis, RT (reverse transcription) capable of cloning with a small amount of plasma was used. Scriptase) -PC
The HCV gene was cloned using the R method.

First, 100 μl of plasma of a single chronic non-A non-B hepatitis patient (# S14) was added to 6 M GTC solution (6 M guanidine thiocyanate, 37.5 mM sodium citrate, 0.75% sarcosyl, 0.2 M). 200 μl of mercaptoethanol) and yeast t-RNA (10 mg / ml) 1
Add μl and stir. Furthermore, 3M sodium acetate (pH
5.2) 20 μl, TE-saturated phenol (pH 7.5-
8.0) 30 μl and chloroform / isoamyl alcohol (49: 1) 70 μl were added and mixed rapidly, stirred for 10 seconds, and then allowed to stand in ice for 15 minutes. 150 in centrifuge
Centrifuge at 00 rpm for 20 minutes at 4 ° C. The aqueous layer is collected, mixed with an equal amount of isopropyl alcohol, and placed at -20 ° C for 1 hour or more. This is centrifuged at 15000 rpm for 20 minutes at 4 ° C. to precipitate. Precipitate the 4M GTC (6M GTC
(Diluted with sterilized water) 100 μl, mixed with an equal amount of isopropyl alcohol, and allowed to stand at −20 ° C. for 1 hour or more. A precipitate is obtained by centrifugation at 15000 rpm for 20 minutes at 4 ° C. After washing with 1 ml of 70% ethanol, it was air-dried at room temperature, dissolved in 10 μl of sterilized water and used as RNA.

For cDNA synthesis, 10 μl of RNA is dispensed into a siliconized tube (0.5 ml), heated at 70 ° C. for 3 minutes, and then rapidly cooled on ice. Next, RNase inhibitor (Takara Shuzo) 1 μl (50 units / μl), dNTP (20 mM each) 1 μl, 100 mM DTT, 5 × RT buffer
(250 mM Tris-HCl (pH 8.5), 375
mM KCl, 15 mM MgCl ₂ ) 4 μl, random oligohexamer primer (100 pmol / μl) 1 μl
l, reverse transcriptase (BRL) (200 units / μl) 1 μl
And add to 20 μl with sterile water. After reacting at 42 ° C for 2 hours, the enzyme was inactivated by heating at 94 ° C for 5 minutes. PCR was performed using this cDNA. PCR detected DN
A two-step method was used to increase the amplification sensitivity and specificity of A. That is, first, the first PCR is applied with two kinds of primers (1st step PCR). Next, it is a method of performing a second PCR using two kinds of primers existing inside the DNA sequence of the PCR product (2nd step PCR).

C6-62 region, C6-66 region, C6-
Primers were synthesized for 4 regions, 79 region and C6-82 region, and used in the two-step method. The PCR primers used are described below. Each region is the sequence reported previously (J. Virol. 65, 1105-1113 (1991); Proc. N.
atl. Acad. Sci. USA 87, 9524-9528 (1990); VirusGen
es 5: 3, 243-259 (1991); J. General Virol. 72, 2697
-2704 (1991)) was set as a reference. The positional relationship between each amplification region and the previously reported sequence (HC-J6) is shown in FIG.

C6-62: 1st PCR primer: (4-1) 5'-ATG GAG ACT AAA CTC ATC AC-
3 '; (6-1) 5'-GAC ATG CAT GTC ATG ATG TA-
3 '; 2nd PCR primer: (4-3) 5'-TAC TTC TAG GAC CGG CCG AT-
3 '; (6-2) 5'-CAC ATT TGA TCC CAC GAT GG-
3 '; C6-79: 1st PCR primer: (6-3) 5'-GGC TAT ACC GGC GAC TTC GA-
3 '; (14-31) 5'-GAT CGG AAG GGA GCT GAG ACC
CGA C-3 '; 2nd PCR primer: (6-4) 5'-GAT CGA CTG TAA CAC ATG TG-
3 '; (14-31) 5'-GAT CGG AAG GGA GCT GAG ACC
CGA C-3 '; C6-66: 1st PCR primer: (6-5) 5'-TGG CTA AAG GAT GTT TGG GA-
3 '; (6-6) 5'-TTC ATC TCC TTG AGC ACG TC-
3 '; 2nd PCR primer: (6-7) 5'-TGG ATA TGC ACG GTG TTG AG-
3 '; (6-8) 5'-CAG GAC TTG CAG TCT GTC AA-
3 '; C6-82: 1st PCR primer: (14-36) 5'-GTC GTC TGC TGC TCA ATG TC-
3 '; (6-9) 5'-AAA AAA AAA AAA AAA TGG CCT-
3 '; 2nd PCR primer: (6-10) 5'-ATC AAT CCG TTG AGC AAC TG-
3 '; (6-11) 5'-ACA GAA AGT AGG AGT AGG CN-
3 '(N represents synthesis by mixing respective bases (G, A, T, C)).

The PCR conditions were as follows: 20 μl of the above cDNA synthesis reaction solution and 10 × PCR buffer (100 mM Tris-HCl (pH 8.3), 500) in a 0.5 ml tube.
mMKCl, 15 mM MgCl ₂ , 0.1% gelatine)
8μl, 2 kinds of 1st step primer (75pmole each),
Add 8 μl of 2 mM dNTPs and make up to 100 μl with sterile water. Heat at 94 ° C for 10 minutes and apply Ampli Taq (Perkin-E
lmer-Cetus) 1 μl (5 units) is added, and after stirring, overlay with mineral oil and gently centrifuge. PCR reaction is modified 94
30 cycles were carried out under the conditions of 1 ° C. for 1 minute, annealing 55 ° C. for 1 minute, and extension 72 ° C. for 2 minutes. Next, in a new 0.5 ml tube, 10 μl of 1st PCR reaction termination solution, 10 × PC
Add 9 µl of R buffer, 2 kinds of 2nd step primers (75 pmole each), 9 µl of 2 mM dNTP, 100 with sterile water
Let μl. Heat at 94 ℃ for 10 minutes, Ampli Taq 1μ
After adding 1 (5 units) and stirring, overlay with mineral oil, gently centrifuge, and perform 2nd PCR under the above conditions. After the reaction, 10 μl of the reaction solution was subjected to agarose gel electrophoresis to detect the specifically amplified DNA fragment.

PCR product (HCV # R6 DNA fragment)
And the determination of the nucleotide sequence It was considered that mutations could be easily introduced into the HCV gene during replication. Therefore, pBR3 is used as a vector to minimize the artificial mutations that occur during cloning.
22 (Sutchliffe, JG, Cold Spring Harbor Sympos
ium, 43, 77-90 (1979)) modified vector (pBM)
Was used. The production of pBM was performed by the procedure shown in FIG. That is, the sequence between the restriction enzyme EcoR V site and the BalI site of pBR322 was deleted with a restriction enzyme,
pUC11 between the coRI site and the Hind III site
9 (Vieria, J., Messing, J., Methods in Enzymolog
y, 153, 3-11 (1987)) from the EcoRI site to the HindIII site of the multiple cloning site (ΔpBR MCS), and then Vsp of pBR322.
The sequence between the I site and the Sca I site was designated as pUC1.
The sequence was replaced with the sequence between the 19 Vsp I site and the Sca I site, and the Pst I site between them was deleted to prepare a pBM vector having a full length of 3122 bp.

The total amount of the PCR reaction solution in which the HCV DNA was detected was mixed with an equal amount of chloroform / isoamyl alcohol (24: 1), the mixture was centrifuged, the aqueous layer was transferred to a 0.5 ml tube, and 1/10 of it was added. Amount of 3M sodium acetate (p
(H5.2) 2 times the amount of ethanol was added and ethanol precipitation was performed. The precipitate is 10 mM Tris-HCl-1 mM EDTA
It was dissolved in 300 μl of (pH 7.4) (TE) and centrifugally filtered with Ultrafree C3TK (Nippon Millipore Limited) to remove residual primer and desalt. The treatment solution was 2 μl of 10 × T4 DNA polymerase buffer solution (30 mM Tris-acetate, 0.66 M potassium acetate, 0.1 M magnesium acetate, 5 mM DTT, 1 mg / ml BSA), 2 μl.
1 μl of mM dNPT and 4 units of T4 DNA polymerase (Takara Shuzo) were added to make 20 μl with sterilized water, and 12 ° C., 1
Reacted for 5 minutes. After the reaction, extraction was performed once each with an equal amount of phenol / chloroform (25:24) and chloroform / isoamyl alcohol (24: 1), and the aqueous layer was ethanol-precipitated. The precipitate was washed with 75% ethanol, air-dried, and then washed with 10x imidazole buffer (0.5M).
Imidazole hydrochloric acid (pH 6.4), 0.18 M magnesium chloride, 50 mM DTT 4 μl, 24% polyethylene glycol 6000 10 μl, 10 mM ATP0.
5 μl and 20 units of T4 DNA kinase (Takara Shuzo) were added to make 40 μl with sterilized water, and the reaction was carried out at 37 ° C. for 1 hour to phosphorylate the 5 ′ end. The enzyme was inactivated by treatment with chloroform / isoamyl alcohol, and the aqueous layer was precipitated with ethanol and washed with 75% ethanol. The DNA of the precipitate was isolated by low melting point agarose gel electrophoresis, extracted twice with TE-saturated phenol, the DNA fragment was precipitated with ethanol, washed with 75% ethanol, and dissolved in 10 μl of sterilized water. Electrophoresis was performed to determine the amount of DNA fragments.

The DNA fragment obtained here was cleaved with a restriction enzyme Sma I in advance, and its 5'end was dephosphorylated by alkaline phosphatase treatment to obtain pBM.
Perform ligation reaction with the vector.

PBM (20 μl) is a restriction enzyme reaction solution 50
μl (10 mM Tris-HCl (pH 8.0), 7 mM
MgCl ₂ , 20 mM KCl, Sma I (Takara Shuzo)
(80 units) at 30 ° C. for 90 minutes, heated at 68 ° C. for 15 minutes, and precipitated with ethanol. The precipitate was washed with 75% ethanol, air-dried, and then washed with 10 × alkaline phosphatase buffer (100 mM Tris-HCl (pH 8.3), 1
5 μl of mM ZnCl ₂ , 10 mM MgCl _{2 and} 1 unit of alkaline phosphatase (derived from cow small intestine; Takara Shuzo) were mixed with sterilized water to make 50 μl, and dephosphorylated by reacting at 37 ° C. for 1 hour. 500 mM EDTA (pH
7.5) Add 0.5 μl, 10% SDS 2.5 μl,
Protease K was further added to a final concentration of 50 μg / ml, the reaction was carried out at 56 ° C. for 30 minutes to inactivate the enzyme, and the vector was isolated by low melting point agarose gel electrophoresis.
Extract twice with TE-saturated phenol, precipitate with ethanol, wash with 75% ethanol, air-dry, and sterilize water 50μ
It was dissolved in 1. 1 μl of the vector was subjected to agarose gel electrophoresis to determine the amount of vector, and the final concentration of Sm was 0.1 μg / ml.
a I cloning vector.

The phosphorylated DNA fragment had a molar ratio of 15 to 2 with respect to 25 ng of Sma I cloning vector.
Add 0 volumes and 10x ligation buffer (0.66M
Tris-HCl (pH 7.6), 50 mM MgCl
₂ , 50 mM DTT) 2 μl, 10 mM hexamine cobalt chloride 2 μl, BSA (1 mg / ml) 2 μl, 10 mM AT
P1 μl and T4 DNA ligase (Takara Shuzo) 350 units were added to make 20 μl with sterilized water, and the ligation reaction was carried out at 16 ° C. overnight. 0.5μ of tRNA (10mg / ml) was added to this reaction mixture.
1 was added and ethanol precipitation was performed, followed by washing with 75% ethanol, the precipitate was dissolved in 10 μl of sterilized water, and half of the amount was used to transform Escherichia coli DH5α strain. A susceptible E. coli strain (competent cell) used for transformation has been reported (J.
Mol. Biol., 166, 577 (1983)) was used.

Transformants were LB-Amp plates (1%
Bactryptone, 0.5% yeast extract, 0.5% sodium chloride, 1.5% agar, ampicillin 50 μg / m
l) After culturing overnight, the colonies appearing on the plate were cultivated in 15 ml tubes containing 3 ml LB-Amp, and 1.5 ml of the culture solution was centrifuged to collect the cells, and minipreparation of plasmid DNA ( Maniatis et al, Mo
leculer Cloning: A Laboratory Manual, 1982) was performed to prepare 15 μl of DNA solution. 2 to 3 μl of each of which are 4 units each of restriction enzymes EcoRI and Hind III, a reaction buffer (50 mM Tris-HCl (pH 7.5), 10
mM MgCl ₂ , 1 mM DTT, 100 mM NaCl)
After reacting in 10 μl at 37 ° C. for 1 hour, agarose gel electrophoresis was performed to confirm the size of the inserted DNA fragment.

Four DNA fragments were confirmed by the above method.

The four kinds of DNA thus obtained were further subjected to Sanger
dideoxy termination method such as r (Science, 214,
1205-1210 (1981)) was used to determine the nucleotide sequence.
In addition, the respective region clones used for the DNA nucleotide sequence determination were C6-62, C6-66, C6-79 and C6.
It was named -82. Also, the determined nucleotide sequence of the gene and the amino acid sequence deduced therefrom are C6-62 as SEQ ID NO: 1, C6-66 as SEQ ID NO: 2, C6-79 as SEQ ID NO: 3, and C6-82 as SEQ ID NO: 4. Indicated. Clone C6-62 has accession number FERM P-13707,
C6-66 is FERM P-13708, C6-79 is FERM P-13709, C6-82 is FERM P
-13710 has been deposited at the Institute of Biotechnology, Institute of Biotechnology, June 28, 1993.

Example 2 Detection of HCV (# R260) Gene by RT-PCR From a single chronic non-A non-B hepatitis patient plasma different from that of Example 1 by the same method as shown in Example 1 above. HCV
RT-PCR of the (# R260) gene was performed to obtain C260
The amplified DNA fragment in the -1 region was detected.

The primers used are shown below.

1st PCR primer: (260-1) 5'-TGA TAG GGT GCT TGC GAG TG
-3 '; (260-2) 5'-GTA GCC GTG GGC GAC CAG TT
C-3 '; 2nd PCR primer: (260-3) 5'-AGA CCG TGC ACC ATG GGC AC
-3 '; (260-4) 5'-TCA TGT CCC ACG CCA TGC GG
TG -3 '.

Cloning of PCR product and determination of nucleotide sequence pUC119 was used as a vector for cloning.
The SmaI site and the gene fragment amplified by PCR using the above primers were cloned by the method shown in Example 1 except that Escherichia coli JM109 strain was used as the transformation host bacterium, and the nucleotide sequence was determined. . Also, the clone used for the determination of the DNA base sequence was named C260-1. The nucleotide sequence of the determined gene and the amino acid sequence deduced therefrom are shown in SEQ ID NO: 5. Clone C260-1 has accession number FERM P-1
It was deposited as 3711 on June 28, 1993 at the Institute of Biotechnology, Institute of Biotechnology, AIST.

Example 3 Expression of HCV (# R260) -derived gene using E. coli a) Construction of expression plasmid Using clone C260-1 DNA as a template, primer 14C-F (5'-CAGAATTCATGA)
GCACAAATCCTAAACCTC-3 ') and primer R-120 (5'-CACTGCAGTTAAA).
PCR reaction was performed using GATTGCGTGATCTATGCC-3 '). PCR reaction is template DN
A 1 μl of 10x PCR buffer (100 mM Tri
s-HCl (pH 8.3), 500 mM KCl, 15
mM MgCl ₂ , 0.1% gelatin) 5μ
l, primers 14C-F, R-120 (each 240p
M), 0.2 μl of 25 mM dNTPs, Taq. p
Olymerase (Boehringer) 0.2μ
1 (1 unit) was added, and the mixture was made up to 50 μl with sterilized water and stirred, and overlaid with mineral oil, denatured at 94 ° C.
Min, annealing 55 ° C. for 0.5 minutes, extension 72 ° C. for 1 minute, and 44 cycles were performed. After the reaction, the total amount of the reaction solution is T
E-Saturated phenol and chloroform treatment, the aqueous layer was ethanol precipitated, dissolved in sterile water, and added with buffer (Takara U
niversal Buffer H) in the restriction enzymes EcoRI and Ps
Digested with tI and agarose gel electrophoresis for about 370
A bp DNA fragment was isolated, treated with TE-saturated phenol and chloroform, and then precipitated with ethanol to purify a DNA fragment of about 370 bp. This purified 370 bp D
The NA fragment was mixed with the vector pKK223-3 previously digested with EcoRI and PstI in the above buffer solution to prepare T4DN.
Ligation reaction was performed with A ligase, and E. coli JM109 was used for transformation.

Transformants were LB-Amp plates (1%
Bactrypton, 0.5% yeast extract, 0.5% Na
Cl, 1.5% agar, 50 μg / ml ampicillin), and the colonies appearing on the plate were cultured in 15 ml tubes containing 3 ml of LB-Amp.
1.5 ml was collected by centrifugation to collect plasmid DNA mini-preparation (Maniatis et al, Moleculer Clon.
ing: A Laboratory Manual, 1982), 20 μl
DNA solution was prepared. 10 μl of the prepared DNA solution was digested with EcoRI and PstI in the above buffer solution and subjected to agarose gel electrophoresis to give a DNA of about 370 bp.
A clone (pR120) in which the fragment was inserted was obtained.

B) Confirmation of expression by Western blotting
And reaction with non-A non-B hepatitis patient serum The above-mentioned E. coli clone was incubated in 3 ml of LB-Amp medium with 37 ml.
After pre-incubating at 3 ° C. for 3 hours, 50 μl of the pre-cultured LB
It was inoculated into 5 ml of Amp medium and cultured at 37 ° C. for 2 hours. IPTG (Wako Pure Chemical Industries, Ltd.) to a final concentration of 2 mM in the culture solution
Was added, and the mixture was further incubated at 37 ° C for 3 hours. 1.5 ml of culture solution
Were collected by centrifugation at 13000 rpm for 2 minutes. 50 μl of TE buffer and 50 μl of 2 × sample buffer (100 mM Tris-HCl (pH
6.8), 20% glycerol, 10% SDS, 5%
2-mercaptoethanol, 0.2% bromophenol blue) was added, and the mixture was suspended and mixed, and the suspension was boiled at 100 ° C. for 5 minutes to prepare a sample.

The above sample was subjected to MINI PROTEAN.
II Dual Slab Cell (Bio-ra
d)) method of Laemmli (Nature, 227, 680
(1970)), and SDS-polyacrylamide gel electrophoresis was performed at 20 mA for 1.5 hours. After migration, cut out the gel and attach PVDF membrane (Millipore) to it.
INI TRANS BLOT Electropho
retic Transfer Cell (Bio-r
Ad) and transferred for 250 mA for 1.75 hours. After the transfer, the membrane was transferred to a buffer solution I ′ containing 5% skimmed milk and 2% BSA (10 mM Na-phosphate).
(PH 7.0), 1% BSA, 0.15M NaCl,
It was immersed in 2.5 mM EDTA) and blocked overnight at 4 ° C. A blocked membrane was placed in a serum sample diluted 40-fold with buffer solution I ′ containing 5% skim milk and 2% BSA, and the mixture was reacted at room temperature for 4 hours. After the reaction, the membrane was replaced with buffer II (10 mM Na-phosphat
e (pH 7.0), 0.15M NaCl, 0.05% T
anti-human IgG-PO diluted with buffer I'containing 2% skim milk to 100 mU / ml after washing 3 times with 20%
The mixture was added to a D-labeled antibody solution (goat antibody) and reacted at room temperature for 30 minutes. After the reaction, remove the membrane and add 5 with Buffer II.
Washed twice. A colored solution (20 mM
Tris-HCl (pH 7.5), 0.5M NaC
1, 0.05% 4-chloro-1-naphthol, 0.01
It was immersed in 8% H ₂ O ₂ and 16.7% methanol) and reacted at room temperature for 30 minutes.

The results are shown in FIG. FIG. 3 shows the results of Western blotting for 5 non-A non-B chronic hepatitis patient sera and 5 healthy human sera. Only the non-A non-B hepatitis patient sera showed a strong positive reaction in all cases. Was detected and the expressed antigen was shown to be useful in the diagnosis of non-A non-B hepatitis patients.

[0064]

INDUSTRIAL APPLICABILITY According to the present invention, HCV which has different homology in the nucleotide sequence and amino acid sequence from the previously reported sequence.
The gene was cloned. The base sequence obtained by the present invention can be expected to be used for nucleic acid diagnosis of non-A non-B hepatitis patients, and the polypeptide prepared based on the base sequence is used for antibody measurement of non-A non-B hepatitis patients. In addition to the above, it can be expected to be applied to an antigen detection system by producing a monoclonal antibody.

[0065]

[Sequence list]

 Sequence number: 1 Sequence length: 1755 Sequence type: Number of nucleic acid strands: Double-stranded topology: Linear array type: cDNA   to   genomic   RNA   Array   AGT   CTT   GAA   GGG   CAG   GGG   TGG   CGG   CTC   CTT   GCT   CCC   ATC   ACG   GCC   TAT   48   Ser   Leu   Glu   Gly   Gln   Gly   Trp   Arg   Leu   Leu   Ala   Pro   Ile   Thr   Ala   Tyr     1   Five   Ten   15   TCC   CAA   CAG   ACG   CGG   GGC   CTA   CTT   GGT   TGC   ATC   ATC   ACT   AGC   CTC   ACA   96   Ser   Gln   Gln   Thr   Arg   Gly   Leu   Leu   Gly   Cys   Ile   Ile   Thr   Ser   Leu   Thr   20   twenty five   30   GGC   CGG   GAC   AAA   AAC   CAA   GTC   GAG   GGG   GAG   GTT   CAA   GTG   GTC   TCC   ACC   144   Gly   Arg   Asp   Lys   Asn   Gln   Val   Glu   Gly   Glu   Val   Gln   Val   Val   Ser   Thr   35   40   45   GCG   ACA   CAA   TCC   TTC   CTG   GCG   ACC   TGC   GTC   AAT   GGC   GCG   TGC   TGG   ACT   192   Ala   Thr   Gln   Ser   Phe   Leu   Ala   Thr   Cys   Val   Asn   Gly   Ala   Cys   Trp   Thr   50   55   60   GTC   TTC   CAT   GGT   GCC   GGC   TCA   AAG   ACC   TTA   GCT   GGC   CCA   AAA   GGT   CCA   240   Val   Phe   His   Gly   Ala   Gly   Ser   Lys   Thr   Leu   Ala   Gly   Pro   Lys   Gly   Pro   65   70   75   80   ATC   ACC   CAG   ATG   TAC   ACT   AAT   GTA   GAC   CTG   GAC   CTC   GTC   GGC   TGG   CAG   288   Ile   Thr   Gln   Met   Tyr   Thr   Asn   Val   Asp   Leu   Asp   Leu   Val   Gly   Trp   Gln   85   90   95   GCG   CCC   CCC   GGG   TCG   CGT   TCT   CTG   ACA   CCA   TGC   ACC   TGC   GGC   AGC   TCA   336   Ala   Pro   Pro   Gly   Ser   Arg   Ser   Leu   Thr   Pro   Cys   Thr   Cys   Gly   Ser   Ser   100   105   110   GAC   CTC   TAT   TTG   GTC   ACG   AGA   CAT   GCT   GAT   GTC   ATT   CCG   GTG   CGC   CGG   384   Asp   Leu   Tyr   Leu   Val   Thr   Arg   His   Ala   Asp   Val   Ile   Pro   Val   Arg   Arg   115   120   125   CGG   GGC   GAC   AGT   AGG   GGA   AGC   CTA   CTC   TCT   CCC   AGA   CCT   GTC   TCC   TAC   432   Arg   Gly   Asp   Ser   Arg   Gly   Ser   Leu   Leu   Ser   Pro   Arg   Pro   Val   Ser   Tyr   130   135   140   TTG   AAA   GGC   TCC   TCG   GGT   GGT   CCG   CTG   CTC   TGC   CCT   TCG   AGG   CAC   GCT   480   Leu   Lys   Gly   Ser   Ser   Gly   Gly   Pro   Leu   Leu   Cys   Pro   Ser   Arg   His   Ala   145   150   155   160   GTG   GGC   ATC   TTC   CGG   GCT   GCT   GTG   TGC   ACC   CGG   GGG   GTT   GCG   AAG   GCG   528   Val   Gly   Ile   Phe   Arg   Ala   Ala   Val   Cys   Thr   Arg   Gly   Val   Ala   Lys   Ala   165   170   175   GTG   GAT   TTC   ATT   CCC   GTT   GAA   TCA   ATG   GAA   ACT   ACT   ATG   CGG   TCT   CCG   576   Val   Asp   Phe   Ile   Pro   Val   Glu   Ser   Met   Glu   Thr   Thr   Met   Arg   Ser   Pro   180   185   190   GTC   TTC   ACG   GAT   AAC   TCA   TCC   CCC   CCG   GCC   GTA   CCG   CAG   ACA   TTC   CAA   624   Val   Phe   Thr   Asp   Asn   Ser   Ser   Pro   Pro   Ala   Val   Pro   Gln   Thr   Phe   Gln   195   200   205   GTG   GCC   CAT   CTA   CAC   GCC   CCT   ACT   GGC   AGC   GGC   AAG   AGC   ACT   AAG   GTG   672   Val   Ala   His   Leu   His   Ala   Pro   Thr   Gly   Ser   Gly   Lys   Ser   Thr   Lys   Val   210   215   220   CCG   GCT   GCA   TAT   GCA   GCC   CAA   GGG   TAT   AAG   GTG   CTC   GTC   CTG   AAC   CCG   720   Pro   Ala   Ala   Tyr   Ala   Ala   Gln   Gly   Tyr   Lys   Val   Leu   Val   Leu   Asn   Pro   225   230   235   240   TCC   GTT   GCC   GCT   ACC   TTG   GGT   TTT   GGG   GCG   TAT   ATG   TCT   AAG   GCA   CAT   768   Ser   Val   Ala   Ala   Thr   Leu   Gly   Phe   Gly   Ala   Tyr   Met   Ser   Lys   Ala   His   245   250   255   GGT   ATC   GAC   CCC   AAC   ATC   AGA   ACT   GGG   GTA   AGG   GCC   ATC   ACC   ACG   GGC   816     Gly   Ile   Asp   Pro   Asn   Ile   Arg   Thr   Gly   Val   Arg   Ala   Ile   Thr   Thr   Gly     260   265   270   GCC   CCT   ATT   ACA   TAC   TCC   ACC   TAC   GGC   AAG   TTC   CTT   GCC   GAC   GGC   GGT   864   Ala   Pro   Ile   Thr   Tyr   Ser   Thr   Tyr   Gly   Lys   Phe   Leu   Ala   Asp   Gly   Gly   275   280   285   TGT   TCC   GGG   GGC   GCC   TAT   GAC   ATC   ATA   ATA   TGT   GAT   GAG   TGC   CAC   TCA   912   Cys   Ser   Gly   Gly   Ala   Tyr   Asp   Ile   Ile   Ile   Cys   Asp   Glu   Cys   His   Ser   290   295   300   ACT   GAC   TCG   ACT   ACC   ATC   TTG   GGC   ATT   GGC   ACA   GTC   CTG   GAC   CAA   GCG   960   Thr   Asp   Ser   Thr   Thr   Ile   Leu   Gly   Ile   Gly   Thr   Val   Leu   Asp   Gln   Ala   305   310   315   320   GAG   ACG   GCT   GGA   GCG   CGG   CTC   GTC   GTG   CTC   GCC   ACC   GCT   ACG   CCT   CCG   1008   Glu   Thr   Ala   Gly   Ala   Arg   Leu   Val   Val   Leu   Ala   Thr   Ala   Thr   Pro   Pro   325   330   335   GGA   TCG   GTC   ACC   GTG   CCA   CAC   CCC   AAT   ATT   GAG   GAG   GTG   GCC   CTG   TCC   1056   Gly   Ser   Val   Thr   Val   Pro   His   Pro   Asn   Ile   Glu   Glu   Val   Ala   Leu   Ser   340   345   350   AAC   GCT   GGA   GAA   ATC   CCC   TTC   TAC   GGC   AAA   GCC   ATC   CCC   ATT   GAG   GTC   1104   Asn   Ala   Gly   Glu   Ile   Pro   Phe   Tyr   Gly   Lys   Ala   Ile   Pro   Ile   Glu   Val   355   360   365   ATC   AAG   GGG   GGA   AGA   CAT   CTC   ATT   TTC   TGC   CAT   TCC   AAG   AAG   AAG   TAT   1152   Ile   Lys   Gly   Gly   Arg   His   Leu   Ile   Phe   Cys   His   Ser   Lys   Lys   Lys   Tyr   370   375   380   GAC   GAG   CTC   GCC   GCA   AAG   CTA   TCA   GCC   CTC   GGA   CTT   AAT   GCT   GTA   GCA   1200   Asp   Glu   Leu   Ala   Ala   Lys   Leu   Ser   Ala   Leu   Gly   Leu   Asn   Ala   Val   Ala   385   390   395   400   TAT   TAT   CGG   GGT   CTT   GAT   GTG   TCC   GTC   ATA   CCG   ACC   AAC   GGA   GAC   GTC   1248   Tyr   Tyr   Arg   Gly   Leu   Asp   Val   Ser   Val   Ile   Pro   Thr   Asn   Gly   Asp   Val   405   410   415   GTT   GTC   GTG   GCA   ACA   GAC   GCT   CTA   ATG   ACG   GGC   TTT   ACC   GGC   GAC   TTT   1296   Val   Val   Val   Ala   Thr   Asp   Ala   Leu   Met   Thr   Gly   Phe   Thr   Gly   Asp   Phe   420   425   430   GAC   TCA   GTG   ATC   GAC   TGT   AAC   ACA   TGT   GTC   ACC   CAG   ACA   GTC   GAT   TTC   1344   Asp   Ser   Val   Ile   Asp   Cys   Asn   Thr   Cys   Val   Thr   Gln   Thr   Val   Asp   Phe   435   440   445   AGC   CTG   GAT   CCC   ACC   TTC   ACC   ATC   GAG   ACG   ACG   ACC   GTG   CCC   CAA   GAC   1392   Ser   Leu   Asp   Pro   Thr   Phe   Thr   Ile   Glu   Thr   Thr   Thr   Val   Pro   Gln   Asp   450   455   460   GCA   GTG   GCG   CGA   TCA   CAG   CGG   CGG   GGT   AGG   ACT   GGT   AGG   GGC   AGG   AGA   1440   Ala   Val   Ala   Arg   Ser   Gln   Arg   Arg   Gly   Arg   Thr   Gly   Arg   Gly   Arg   Arg   465   470   475   480   GGC   ATC   TAC   AGG   TTT   GTG   ACT   CCA   GGA   GAA   CGG   CCC   TCG   GGC   ATG   TTC   1488   Gly   Ile   Tyr   Arg   Phe   Val   Thr   Pro   Gly   Glu   Arg   Pro   Ser   Gly   Met   Phe   485   490   495   GAT   TCC   TCG   GTC   CTG   TGT   GAG   TGC   TAT   GAC   GCG   GGC   TGT   GCT   TGG   TAC   1536   Asp   Ser   Ser   Val   Leu   Cys   Glu   Cys   Tyr   Asp   Ala   Gly   Cys   Ala   Trp   Tyr   500   505   510   GAG   CTC   ACG   CCT   GCT   GAG   ACC   TCG   GTT   AGG   TTG   CGG   GCT   TAC   CTG   AAT   1584   Glu   Leu   Thr   Pro   Ala   Glu   Thr   Ser   Val   Arg   Leu   Arg   Ala   Tyr   Leu   Asn   515   520   525   ACA   CCA   GGG   TTG   CCC   GTC   TGC   CAG   GAC   CAT   CTG   GAG   TTT   TGG   GAG   AGC   1632   Thr   Pro   Gly   Leu   Pro   Val   Cys   Gln   Asp   His   Leu   Glu   Phe   Trp   Glu   Ser   530   535   540   GTC   TCC   ACA   GGC   CTC   ACC   CAC   ATA   GAT   GCC   CAC   TTC   CTG   TCC   CAG   ACT   1680   Val   Ser   Thr   Gly   Leu   Thr   His   Ile   Asp   Ala   His   Phe   Leu   Ser   Gln   Thr   545   550   555   560   AAG   CAG   GCA   GGA   GAC   AAC   TTC   CCC   TAC   CTG   GTA   GCA   TAT   CAA   GCC   ACA   1728   Lys   Gln   Ala   Gly   Asp   Asn   Phe   Pro   Tyr   Leu   Val   Ala   Tyr   Gln   Ala   Thr   565   570   575   GTG   TGC   GCC   AGG   GCA   CAA   GCT   CCA   CCT   1755   Val   Cys   Ala   Arg   Ala   Gln   Ala   Pro   Pro   580   585     Sequence number: 2 Array length: 1451 Array type: Number of nucleic acid strands: Double-stranded topology: Linear array type: cDNA to genomic RNA Sequence T GAC TTC AAG ACC TGG CTC CAG TCC AAG CTC CTG CCG CGG TTA CCG 46 Asp Phe Lys Thr Trp Leu Gln Ser Lys Leu Leu Pro Arg Leu Pro 1 5 10 15 GGG GTC CCT TTT TTC TCG TGT CAA CGC GGG TAC AAG GGA GTC TGG CGG 94 Gly Val Pro Phe Phe Ser Cys Gln Arg Gly Tyr Lys Gly Val Trp Arg 20 25 30 GGG GAC GGT ATC ATG CAG ACC ACC TGC CCG TGT GGA GCA CAG ATC ACC 142 Gly Asp Gly Ile Met Gln Thr Thr Cys Pro Cys Gly Ala Gln Ile Thr 35 40 45 GGA CAT GTC AAA AAC GGT TCC ATG AGG ATC GTT GGG CCT AAA GCC TGC 190 Gly His Val Lys Asn Gly Ser Met Arg Ile Val Gly Pro Lys Ala Cys 50 55 60 AGC AAC ACG TGG CAT GGA ACG TTC CCC ATC AAC GCA TAC ACC ACG GGC 238 Ser Asn Thr Trp His Gly Thr Phe Pro Ile Asn Ala Tyr Thr Thr Gly 65 70 75 CCA TGC GCA CCC TCC CCG GCG CCA AAC TAT TCC AGG GCG CTA TGG CGG 286 Pro Cys Ala Pro Ser Pro Ala Pro Asn Tyr Ser Arg Ala Leu Trp Arg 80 85 90 95 GTG GCC GCT GAG GAG TAC GTG GAG GTT ACG CGG GTG GGG GAT TTC CAC 334 Val Ala Ala Glu Glu Tyr Val Glu Val Thr Arg Val Gly Asp Phe His 100 105 110 TAC GTG ACG GGC ATG ACC ACT GAC AAC GTA AAG TGC CCA TGC CAG GTT 382 Tyr Val Thr Gly Met Thr Thr Asp Asn Val Lys Cys Pro Cys Gln Val 115 120 125 CCG GCC CCT GAA TTC TTC ACT GAG GTG GAT GGA GTG CGG TTG CAC AGG 430 Pro Ala Pro Glu Phe Phe Thr Glu Val Asp Gly Val Arg Leu His Arg 130 135 140 TAC GCT CCG GCG TGC AAA CCC CTC CTA CGG GAG GAG GTC ACA TTC CAG 478 Tyr Ala Pro Ala Cys Lys Pro Leu Leu Arg Glu Glu Val Thr Phe Gln 145 150 155 GTT GGG CTC AAC CAA TAC CTG GTT GGG TCA CAG CTC CCA TGC GAG CCC 526 Val Gly Leu Asn Gln Tyr Leu Val Gly Ser Gln Leu Pro Cys Glu Pro 160 165 170 175 GAA CCG GAT GTA GCA GTG CTA ACT TCC ATG CTT ACC GAC CCC TCC CAC 574 Glu Pro Asp Val Ala Val Leu Thr Ser Met Leu Thr Asp Pro Ser His 180 185 190 ATC ACA GCA GAG ACG GCA AAG CGT AGG CTG GCT AGG GGG TCT CCC CCC 622 Ile Thr Ala Glu Thr Ala Lys Arg Arg Leu Ala Arg Gly Ser Pro Pro 195 200 205 TCC TTG GCC AGT TCT TCA GCT AGC CAG TTA TCT GCG CCT TCC TTG AAG 670 Ser Leu Ala Ser Ser Ser Ala Ser Gln Leu Ser Ala Pr o Ser Leu Lys 210 215 220 GCG ACA TGC ACT ACC CAT CAT GAC TCC CCG GAC GTT GAC CTC ATC GAG 718 Ala Thr Cys Thr Thr His His Asp Ser Pro Asp Val Asp Leu Ile Glu 225 230 235 GCC AAC CTC CTG TGG CGG CAG GAG ATG GGC GGG AAC ATC ACC CGC GTG 766 Ala Asn Leu Leu Trp Arg Gln Glu Met Gly Gly Asn Ile Thr Arg Val 240 245 250 255 GAG TCA GAG AAT AAG GTA GTA ATT TTG GAC TCT TTC GAT CCG CTC CGA 814 Glu Ser Glu Asn Lys Val Val Ile Leu Asp Ser Phe Asp Pro Leu Arg 260 265 270 GCG GAG GAG GAC GAG AGG GAA CCA TCC GTT GCG GCG GAG ATC TTG CGG 862 Ala Glu Glu Asp Glu Arg Glu Pro Ser Val Ala Ala Glu Ile Leu Arg 275 280 285 AAA ACC AAG AGG TTC CCC CCG GCG ATG CCC ATA TGG GCA CGC CCG GAT 910 Lys Thr Lys Arg Phe Pro Pro Ala Met Pro Ile Trp Ala Arg Pro Asp 290 295 300 TAC AAC CCT CCG TTG CTA GAG TCC TGG AAA GAC CCG GAC TAC GTC CCT 958 Tyr Asn Pro Pro Leu Leu Glu Ser Trp Lys Asp Pro Asp Tyr Val Pro 305 310 315 CCG GTG GTA CAC GGG TGC CCG CTA CCA CCT ACC AAA GCT CCT CCG ATA 1006 Pro Val Val His Gly Cys Pro Leu Pro P ro Thr Lys Ala Pro Pro Ile 320 325 330 335 CCA CCC CCA CGG AGA AAG AGG ACG GTA GTC CTG ACA GAG TCC ACT GTG 1054 Pro Pro Pro Arg Arg Lys Arg Thr Val Val Leu Thr Glu Ser Thr Val 340 345 350 TCT TCT GCC TTG GCG GAG CTT GCT ACT AAG ACC TTT GGC AGC TCC GGG 1102 Ser Ser Ala Leu Ala Glu Leu Ala Thr Lys Thr Phe Gly Ser Ser Gly 355 360 365 TCG TCG GCC GTC GAC AGC GGC ACA GCG ACT GCT CCT CCC GAC CAG GCC 1150 Ser Ser Ala Val Asp Ser Gly Thr Ala Thr Ala Pro Pro Asp Gln Ala 370 375 380 TCC GCC GAC GGC GAC CAA GGA TCT GAC GTT GAG TCG TAT TCC TCT ATG 1198 Ser Ala Asp Gly Asp Gln Gly Ser Asp Val Glu Ser Tyr Ser Ser Met 385 390 395 CCC CCT CTT GAG GGA GAG CCG GGG GAC CCC GAT CTC AGC GAC GGG TCT 1246 Pro Pro Leu Glu Gly Glu Pro Gly Asp Pro Asp Leu Ser Asp Gly Ser 400 405 410 415 TGG TCT ACT GTG AGC GAG GAG GCT GGT GAG GAC GTC GTC TGC TGC TCA 1294 Trp Ser Thr Val Ser Glu Glu Ala Gly Glu Asp Val Val Cys Cys Ser 420 425 430 ATG TCC TAC ACA TGG ACA GGC GCC CTG ATC ACG CCA TGC GCT GCG GAG 1342 Met Ser Tyr T hr Trp Thr Gly Ala Leu Ile Thr Pro Cys Ala Ala Glu 435 440 445 GAA AGC AAG CTG CCC ATC AAC CCG TTG AGC AAC TCT TTG TTG CGT CAC 1390 Glu Ser Lys Leu Pro Ile Asn Pro Leu Ser Asn Ser Leu Leu Arg His 450 455 460 CAC AAC ATG GTC TAC GCT ACA ACA TCC CGC AGC GCA AGC TTA CGG CAG 1438 His Asn Met Val Tyr Ala Thr Thr Ser Arg Ser Ala Ser Leu Arg Gln 465 470 475 AAG AAA GCC ACT T 1451 Lys Lys Ala Thr 480 Sequence number: 3 Sequence length: 2093 Sequence type: Number of nucleic acid strands: Double-stranded topology: Linear array type: cDNA   to   genomic   RNA   Array   TC   ACC   CAG   ACA   GTC   GAT   TTT   AGC   TTG   GAT   CCC   ACC   TTC   ACC   ATT   GAC   47   Thr   Gln   Thr   Val   Asp   Phe   Ser   Leu   Asp   Pro   Thr   Phe   Thr   Ile   Asp   1   Five   Ten   15   ACG   ACG   ACC   GTG   CCC   CAA   GAC   GCA   GTG   TCG   CGC   TCA   CAG   CGG   CGG   GGC   95   Thr   Thr   Thr   Val   Pro   Gln   Asp   Ala   Val   Ser   Arg   Ser   Gln   Arg   Arg   Gly   20   twenty five   30   AGG   ACT   GGC   AGG   GGC   AGG   AGA   GGC   ATC   TAC   AGG   TTT   GTG   ACT   CCA   GGA   143   Arg   Thr   Gly   Arg   Gly   Arg   Arg   Gly   Ile   Tyr   Arg   Phe   Val   Thr   Pro   Gly   35   40   45   GAA   CGG   CCT   TCG   GGC   ATG   TTC   GAT   TCT   TCC   GTC   CTG   TGT   GAG   TGC   TAT   191   Glu   Arg   Pro   Ser   Gly   Met   Phe   Asp   Ser   Ser   Val   Leu   Cys   Glu   Cys   Tyr   50   55   60   GAC   GCG   GGC   TGT   GCT   TGG   TAT   GAG   CTC   ACG   CCT   GCT   GAG   ACT   TCA   GTT   239   Asp   Ala   Gly   Cys   Ala   Trp   Tyr   Glu   Leu   Thr   Pro   Ala   Glu   Thr   Ser   Val   65   70   75   AGG   TTG   CGG   GCT   TAC   CTG   AAT   ACA   CCA   GGG   TTG   CCC   GTC   TGC   CAG   GAC   287   Arg   Leu   Arg   Ala   Tyr   Leu   Asn   Thr   Pro   Gly   Leu   Pro   Val   Cys   Gln   Asp   80   85   90   95   CAT   CTG   GAG   TTT   TGG   GAG   AGC   GTC   TTC   ACA   GGC   CTC   ACC   CAC   ATA   GAT   335   His   Leu   Glu   Phe   Trp   Glu   Ser   Val   Phe   Thr   Gly   Leu   Thr   His   Ile   Asp   100   105   110   GCC   CAT   TTT   CTG   TCC   CAG   ACT   AAA   CAG   GCA   GGA   GAC   AAC   TTC   CCC   TAC   383   Ala   His   Phe   Leu   Ser   Gln   Thr   Lys   Gln   Ala   Gly   Asp   Asn   Phe   Pro   Tyr   115   120   125   CTG   GTA   GCA   TAC   CAA   GCC   ACA   GTG   TGC   GCC   AGA   GCT   CAA   GCT   CCA   CCT   431   Leu   Val   Ala   Tyr   Gln   Ala   Thr   Val   Cys   Ala   Arg   Ala   Gln   Ala   Pro   Pro   130   135   140   CCA   TCA   TGG   GAT   CAA   ATG   TGG   AAG   TGT   CTC   ATA   CGG   CTC   AAA   CCC   ACG   479   Pro   Ser   Trp   Asp   Gln   Met   Trp   Lys   Cys   Leu   Ile   Arg   Leu   Lys   Pro   Thr   145   150   155   CTG   CAC   GGG   CCA   ACA   CCC   CTG   CTG   TAT   AGG   CTA   GGA   GCC   GTC   CAA   AAT   527   Leu   His   Gly   Pro   Thr   Pro   Leu   Leu   Tyr   Arg   Leu   Gly   Ala   Val   Gln   Asn   160   165   170   175   GAG   ATC   ACC   CTC   ACA   CAC   CCC   ATG   ACC   AAA   TTC   ATC   ATG   GCA   TGC   ATG   575   Glu   Ile   Thr   Leu   Thr   His   Pro   Met   Thr   Lys   Phe   Ile   Met   Ala   Cys   Met   180   185   190     TCG   GCT   GAC   CTG   GAG   GTC   GTT   ACT   AGC   ACC   TGG   GTG   CTA   GTA   GGC   GGA   623   Ser   Ala   Asp   Leu   Glu   Val   Val   Thr   Ser   Thr   Trp   Val   Leu   Val   Gly   Gly   195   200   205   GTC   CTT   GCA   GCT   CTG   GCT   GCA   TAT   TGC   TTG   ACA   ACA   GGC   AGT   GTG   GTC   671   Val   Leu   Ala   Ala   Leu   Ala   Ala   Tyr   Cys   Leu   Thr   Thr   Gly   Ser   Val   Val   210   215   220   ATT   GTG   GGT   AGG   ATC   ATC   TTG   TCC   GGG   AGG   CCG   GCT   GTT   ATT   CCC   GAC   719   Ile   Val   Gly   Arg   Ile   Ile   Leu   Ser   Gly   Arg   Pro   Ala   Val   Ile   Pro   Asp   225   230   235   AGG   GAA   GTC   CTC   TAC   CGG   GAG   TTC   GAT   GAG   ATG   GAA   GAG   TGC   GCC   TCA   767   Arg   Glu   Val   Leu   Tyr   Arg   Glu   Phe   Asp   Glu   Met   Glu   Glu   Cys   Ala   Ser   240   245   250   255   CAC   CTC   CCT   TAC   ATC   GAA   CAG   GGA   ATG   CAG   CTT   GCC   GAG   CAA   TTC   AAG   815   His   Leu   Pro   Tyr   Ile   Glu   Gln   Gly   Met   Gln   Leu   Ala   Glu   Gln   Phe   Lys   260   265   270   CAG   AAG   GCG   CTC   GGA   TTG   CTG   CAA   ACA   GCC   ACC   AAG   CAC   GCG   GAG   GCT   863   Gln   Lys   Ala   Leu   Gly   Leu   Leu   Gln   Thr   Ala   Thr   Lys   His   Ala   Glu   Ala   275   280   285   GCT   GCT   CCC   GTG   GTA   GAA   TCC   AAG   TGG   CGA   GCC   CTT   GAG   ACC   TTC   TGG   911   Ala   Ala   Pro   Val   Val   Glu   Ser   Lys   Trp   Arg   Ala   Leu   Glu   Thr   Phe   Trp   290   295   300   GCG   AAG   GAC   ATG   TGG   AAT   TTC   ATC   AGC   GGG   ATA   CAG   TAC   CTA   GCA   GGC   959   Ala   Lys   Asp   Met   Trp   Asn   Phe   Ile   Ser   Gly   Ile   Gln   Tyr   Leu   Ala   Gly   305   310   315   TTG   TCC   ACT   CTG   CCT   GGG   AAC   CCC   GCG   ATA   GCA   TCA   CTG   ATG   GCA   TTC   1007   Leu   Ser   Thr   Leu   Pro   Gly   Asn   Pro   Ala   Ile   Ala   Ser   Leu   Met   Ala   Phe   320   325   330   335   ACA   GCC   TCT   ATC   ACC   AGC   CCG   CTC   TCC   ACC   CAG   AAT   ACC   CTA   TTA   TTT   1055   Thr   Ala   Ser   Ile   Thr   Ser   Pro   Leu   Ser   Thr   Gln   Asn   Thr   Leu   Leu   Phe   340   345   350   AAC   ATC   TGG   GGG   GGA   TGG   GTG   GCT   GCC   CAA   CTC   GCC   CCC   CCC   AGT   GCT   1103   Asn   Ile   Trp   Gly   Gly   Trp   Val   Ala   Ala   Gln   Leu   Ala   Pro   Pro   Ser   Ala   355   360   365   GCT   TCG   GCT   TTC   GTG   GGC   GCC   GGT   ATC   GCC   GGT   GCG   GCT   GTC   GGC   AGC   1151   Ala   Ser   Ala   Phe   Val   Gly   Ala   Gly   Ile   Ala   Gly   Ala   Ala   Val   Gly   Ser   370   375   380   ATA   GGT   CTT   GGG   AAG   GTG   CTT   GTG   GAC   ATC   TTG   GCG   GGA   TAT   GGG   GCA   1199   Ile   Gly   Leu   Gly   Lys   Val   Leu   Val   Asp   Ile   Leu   Ala   Gly   Tyr   Gly   Ala   385   390   395   GGG   GTG   GCT   GGC   GCG   TTC   GTA   GCT   TTT   AAG   ATC   ATG   AGC   GGC   GAG   GTT   1247   Gly   Val   Ala   Gly   Ala   Phe   Val   Ala   Phe   Lys   Ile   Met   Ser   Gly   Glu   Val   400   405   410   415   GCC   TTC   ACA   GAG   GAC   CTG   GTT   AAC   TTA   CTC   CCT   GCC   ATC   CTC   TCT   CCC   1295   Ala   Phe   Thr   Glu   Asp   Leu   Val   Asn   Leu   Leu   Pro   Ala   Ile   Leu   Ser   Pro   420   425   430   GGC   GCC   CTA   GTC   GTC   GGG   GTC   GTG   TGC   GCA   GCA   ATA   CTG   CGT   CGG   CAC   1343   Gly   Ala   Leu   Val   Val   Gly   Val   Val   Cys   Ala   Ala   Ile   Leu   Arg   Arg   His   435   440   445   GTG   GGC   CCG   GGA   GAG   GGG   GCT   GTA   CAG   TGG   ATG   AAC   CGG   CTG   ATA   GCG   1391   Val   Gly   Pro   Gly   Glu   Gly   Ala   Val   Gln   Trp   Met   Asn   Arg   Leu   Ile   Ala     450   455   460   TTC   GCC   TCG   CGG   GGT   AAC   CAC   GTT   TCC   CCC   GCG   CAC   TAT   GTG   CCT   GAG   1439   Phe   Ala   Ser   Arg   Gly   Asn   His   Val   Ser   Pro   Ala   His   Tyr   Val   Pro   Glu   465   470   475   AGC   GAC   GCT   GCG   GCG   CGT   GTT   ACT   CAG   ATC   CTC   TCC   GGC   CTT   ACC   ATC   1487   Ser   Asp   Ala   Ala   Ala   Arg   Val   Thr   Gln   Ile   Leu   Ser   Gly   Leu   Thr   Ile   480   485   490   495   ACT   CAG   CTG   CTG   AAG   AGG   CTT   CAC   CAC   TGG   ATC   AAT   GAG   GAC   TGC   TCC   1535   Thr   Gln   Leu   Leu   Lys   Arg   Leu   His   His   Trp   Ile   Asn   Glu   Asp   Cys   Ser   500   505   510   ACG   CCA   TGC   TCC   GGT   TCG   TGG   CTA   AGG   GAT   GTT   TGG   GAC   TGG   ATA   TGC   1583   Thr   Pro   Cys   Ser   Gly   Ser   Trp   Leu   Arg   Asp   Val   Trp   Asp   Trp   Ile   Cys   515   520   525   ACG   GTG   TTG   ACT   GAC   TTC   AAG   ACC   TGG   CTC   CAG   TCC   AAG   CTC   CTG   CCG   1631   Thr   Val   Leu   Thr   Asp   Phe   Lys   Thr   Trp   Leu   Gln   Ser   Lys   Leu   Leu   Pro   530   535   540   CGG   TTA   CCG   GGG   GTC   CCT   TTC   TTC   TCG   TGT   CAA   CGC   GGG   TAC   AAG   GGA   1679   Arg   Leu   Pro   Gly   Val   Pro   Phe   Phe   Ser   Cys   Gln   Arg   Gly   Tyr   Lys   Gly   545   550   555   GTC   TGG   CGG   GGG   GAC   GGT   ATC   ATG   CAG   ACC   ACC   TGC   CCG   TGT   GGA   GCA   1727   Val   Trp   Arg   Gly   Asp   Gly   Ile   Met   Gln   Thr   Thr   Cys   Pro   Cys   Gly   Ala   560   565   570   575   CAG   ATC   ACC   GGA   CAT   GTC   AAA   AAC   GGT   TCC   ATG   AGG   ATC   GTC   GGG   CCT   1775   Gln   Ile   Thr   Gly   His   Val   Lys   Asn   Gly   Ser   Met   Arg   Ile   Val   Gly   Pro   580   585   590   AAA   ACC   TGC   AGC   AGC   ACG   TGG   CAT   GGA   ACG   TTC   CCC   ATC   AAC   GCA   TAC   1823   Lys   Thr   Cys   Ser   Ser   Thr   Trp   His   Gly   Thr   Phe   Pro   Ile   Asn   Ala   Tyr   595   600   605   ACC   ACA   GGC   CCA   TGC   GCA   CCC   TCC   CCG   GCG   CCA   AAC   TAT   TCC   AGG   GCG   1871   Thr   Thr   Gly   Pro   Cys   Ala   Pro   Ser   Pro   Ala   Pro   Asn   Tyr   Ser   Arg   Ala   610   615   620   CTA   TGG   CGG   GTG   GCC   GCT   GAG   GAG   TAC   GTG   GAG   GTT   ACG   CGG   GTG   GGG   1919   Leu   Trp   Arg   Val   Ala   Ala   Glu   Glu   Tyr   Val   Glu   Val   Thr   Arg   Val   Gly   625   630   635   GAT   TTC   CAC   TAC   GTG   ACG   GGC   ATG   ACC   ACT   GAC   AAC   GTA   AAG   TGC   CCA   1967   Asp   Phe   His   Tyr   Val   Thr   Gly   Met   Thr   Thr   Asp   Asn   Val   Lys   Cys   Pro   640   645   650   655   TGC   CAG   GTT   CCG   GCC   CCT   GAA   TTC   TTC   ACT   GAG   GTG   GAT   GGA   GTG   CGG   2015   Cys   Gln   Val   Pro   Ala   Pro   Glu   Phe   Phe   Thr   Glu   Val   Asp   Gly   Val   Arg   660   665   670   CTG   CAC   AGG   TAC   GCT   CCG   GCG   TGC   AAA   CCC   CTC   CTA   CGG   GAG   GAG   GTC   2063   Leu   His   Arg   Tyr   Ala   Pro   Ala   Cys   Lys   Pro   Leu   Leu   Arg   Glu   Glu   Val   675   680   685   ACA   TTC   CAG   GTT   AGG   CTC   AAC   CAA   TAC   CTG   2093   Thr   Phe   Gln   Val   Arg   Leu   Asn   Gln   Tyr   Leu   690   695     Sequence number: 4 Sequence length: 1386 Sequence Type: Number of nucleic acid strands: Double-stranded topology: Linear array type: cDNA to genomic RNA sequence AGC AAG GCC GTT AAC CAC ATC CGC TCC GTG TGG AAG GAC TTG CTG GAA 48 Ser Lys Ala Val Asn His Ile Arg Ser Val Trp Lys Asp Leu Leu Glu 1 5 10 15 GAC AAT GTA ACA CCA GTT GAC ACC ACC GTC ATG GCA AAA AGT GAG GTT 96 Asp Asn Val Thr Pro Val Asp Thr Thr Val Met Ala Lys Ser Glu Val 20 25 30 TTC TGC GTC CAA CCA GAG AAA GGA GGC CGC AAG CCA GCT CGC CTT ATC 144 Phe Cys Val Gln Pro Glu Lys Gly Gly Arg Lys Pro Ala Arg Leu Ile 35 40 45 GTA TTC CCA GAC TTG GGA GTT CGT GTG TAC GAG AAG ATG GCC CTT TAT 192 Val Phe Pro Asp Leu Gly Val Arg Val Tyr Glu Lys Met Ala Leu Tyr 50 55 60 AAT GTG GTC TCC ACC CTT CCT CAG GCC GTG ATG GGC TCC TCA TAC GGA 240 Asn Val Val Ser Thr Leu Pro Gln Ala Val Met Gly Ser Ser Tyr Gly 65 70 75 80 CTC CAA TAC TCC CCT GGG CAG CGG GTC GAG TTC CTG GTG AAT GCC TGG 288 Leu Gln Tyr Ser Pro Gly Gln Arg Val Glu Phe Leu Val Asn Ala Trp 85 90 95 AAG TCA AAG AAA TGC CCT ATG GGC TTC TCA TAT GAC AAC CGC TGC TTT 336 Lys Ser Lys Lys Cys Pro Met Gly Phe Ser Tyr Asp Asn Arg C ys Phe 100 105 110 GAC TCG ACG GTC ACT GAG AGT GAC ATC CGT ATT GAG GAG TCA ATT TAC 384 Asp Ser Thr Val Thr Glu Ser Asp Ile Arg Ile Glu Glu Ser Ile Tyr 115 120 125 CAA TGT TGT GAC CTG GCC CCC GAA GCC AGA CAG GCC ATA AAG TCG CTC 432 Gln Cys Cys Asp Leu Ala Pro Glu Ala Arg Gln Ala Ile Lys Ser Leu 130 135 140 ACA GAG CGG CTC TAC ATC GGG GGT CCC CTG ACC AAT TCA AAA GGG CAG 480 Thr Glu Arg Leu Tyr Ile Gly Gly Pro Leu Thr Asn Ser Lys Gly Gln 145 150 155 160 AAC TGC GGC TAC CGC CGG TGC CGC GCG AGT GGC GTG CTG ACG ACT AGC 528 Asn Cys Gly Tyr Arg Arg Cys Arg Ala Ser Gly Val Leu Thr Thr Ser 165 170 175 TGC GGT AAT ACC CTT ACA TGT TAC TTG AAG GCC TCT GCA GCC TGT CGA 576 Cys Gly Asn Thr Leu Thr Cys Tyr Leu Lys Ala Ser Ala Ala Cys Arg 180 185 190 GCT GCA AAG CTC CGG GAC TGC ACG ATG CTC GTG AAC GGA GAC GAC CTT 624 Ala Ala Lys Leu Arg Asp Cys Thr Met Leu Val Asn Gly Asp Asp Leu 195 200 205 GTC GTT ATC TGT GAA AGC GCG GGA ACC CAA GAG GAT GAG GCG AAC CTA 672 Val Val Ile Cys Glu Ser Ala Gly Thr Gln Glu A sp Glu Ala Asn Leu 210 215 220 CGA GTC TTC ACG GAG GCT ATG ACT AGG TAC TCT GCC CCC CCC GGG GAC 720 Arg Val Phe Thr Glu Ala Met Thr Arg Tyr Ser Ala Pro Pro Gly Asp 225 230 235 240 CCG CCC CGA CCA GAA TAC GAC TTG GAG CTG ATA ACA TCA TGC TCC TCC 768 Pro Pro Arg Pro Glu Tyr Asp Leu Glu Leu Ile Thr Ser Cys Ser Ser 245 250 255 AAT GTG TCG GTC GCG CAC GAT GCA TCT GGC AAA AGG GTG TAC TAC CTC 816 Asn Val Ser Val Ala His Asp Ala Ser Gly Lys Arg Val Tyr Tyr Leu 260 265 270 ACC CGC GAC CCC TCC ACC CCC CTC GCA CGA GCT GCG TGG GAG ACA GCT 864 Thr Arg Asp Pro Ser Thr Pro Leu Ala Arg Ala Ala Trp Glu Thr Ala 275 280 285 AGA CAC ACT CCA GTA AAC TCC TGG CTA GGC AAC ATC ATT ATG TAT GCG 912 Arg His Thr Pro Val Asn Ser Trp Leu Gly Asn Ile Ile Met Tyr Ala 290 295 300 CCC ACC TTA TGG GCA AGG ATG ATT CTA ATG ACT CAT TTC TTC TCC ATC 960 Pro Thr Leu Trp Ala Arg Met Ile Leu Met Thr His Phe Phe Ser Ile 305 310 315 320 CTT CTA GCT CAG GAG CAA CTT GAA AAA GCC CTG GAT TGT CAG ATC TAC 1008 Leu Leu Ala Gln Glu Gln Leu Glu Lys Ala Leu Asp Cys Gln Ile Tyr 325 330 335 GGG GCC TGT TAC TCC ATT GAG CCA CTT GAC CTA CCT CAG ATC ATT GAA 1056 Gly Ala Cys Tyr Ser Ile Glu Pro Leu Asp Leu Pro Gln Ile Ile Glu 340 345 350 CGA CTC CAT GGT CTT AGC GCA TTT TCA CTC CAT AGT TAC TCT CCA GGT 1104 Arg Leu His Gly Leu Ser Ala Phe Ser Leu His Ser Tyr Ser Pro Gly 355 360 365 GAG ATC AAT AGG GTG GCT TCA TGC CTC AGG AAA CTT GGG GTA CCG CCC 1152 Glu Ile Asn Arg Val Ala Ser Cys Leu Arg Lys Leu Gly Val Pro Pro 370 375 380 TTG CGA GTC TGG AGA CAT CGG GCC AGA AGT GTC CGC GCT AAG CTA CTG 1200 Leu Arg Val Trp Arg His Arg Ala Arg Ser Val Arg Ala Lys Leu Leu 385 390 395 400 TCC CAG GGG GGG AGG GCT GCT ACT TGT GGC AAG TAC CTC TTC AAC TGG 1248 Ser Gln Gly Gly Arg Ala Ala Thr Cys Gly Lys Tyr Leu Phe Asn Trp 405 410 415 GCA GTA AGG ACC AAG CTC AAA CTC ACT CCA ATC CCG GCT GCG TCC CAG 1296 Ala Val Arg Thr Lys Leu Lys Leu Thr Pro Ile Pro Ala Ala Ser Gln 420 425 430 TTG GAC TTG TCC AGC TGG TTC CTG GCT GGT TAC AGC GGG GAA CAC ATA 1344 Leu Asp Leu Ser Ser Trp Phe Leu Ala Gly Tyr Ser Gly Glu His Ile 435 440 445 TAT CAC AGC CTG TCT CGT GCC CGA CCC CGC TGG TTC ATG TTG 1386 Tyr His Ser Leu Ser Arg Ala Arg Pro Arg Trp Phe Met Leu 450 455 460 Sequence number: 5 Sequence length: 937 Sequence type: Number of nucleic acid strands: Double-stranded topology: Linear array type: cDNA to genomic RNA Sequence A AAT CCT AAA CCT CAA AGA AAA ACC AAA AGA AAC GCA AAC CGC CGC 46 Asn Pro Lys Pro Gln Arg Lys Thr Lys Arg Asn Ala Asn Arg Arg 1 5 10 15 CCA CAG GAC GTC AAG TTC CCG GGT GGC GGC CAG ATC GTT GGC GGA GTT 94 Pro Gln Asp Val Lys Phe Pro Gly Gly Gly Gln Ile Val Gly Gly Val 20 25 30 TAC TTG CTG CCG CGC AGG GGC CCC AGG TTG GGT GTG CGC GCG ACG AGG 142 Tyr Leu Leu Pro Arg Arg Gly Pro Arg Leu Gly Val Arg Ala Thr Arg 35 40 45 AAG ACT TCC GAG CGA TCC CAG CCG CGT GGG AGG CGC CAG CCC ATC CCG 190 Lys Thr Ser Glu Arg Ser Gln Pro Arg Gly Arg Arg Gln Pro Ile Pro 50 55 60 AAA GAT CGG CGC TCC ACC GGC AAG TCC TGG GGA AAG CCA GGA TAT CCT 238 Lys Asp Arg Arg Ser Thr Gly Lys Ser Trp Gly Lys Pro Gly Tyr Pro 65 70 75 TGG CCT CTG TAT GGA AAC GAG GGC TGC GGC TGG GCA GGT TGG CTC CTG 286 Trp Pro Leu Tyr Gly Asn Glu Gly Cys Gly Trp Ala Gly Trp Leu Leu 80 85 90 95 TCC CCC CGC GGG TCT CGT CCT ACT TGG GGC CCC ACT GAC CCC CGG CAT 334 Ser Pro Arg Gly Ser Arg Pro Thr Trp Gly Pro Thr Asp Pro Arg Hi s 100 105 110 AGA TCA CGC AAT CTT GGT AAG GTC ATC GAC ACC ATT ACG TGT GGT TTT 382 Arg Ser Arg Asn Leu Gly Lys Val Ile Asp Thr Ile Thr Cys Gly Phe 115 120 125 GCC GAC CTC ATG GGG TAC ATC CCT GTC GTT GGC GCC CCG GTC GGA GGC 430 Ala Asp Leu Met Gly Tyr Ile Pro Val Val Gly Ala Pro Val Gly Gly 130 135 140 GTC GCC AGA GCC TTG GCA CAC GGT GTT AGG GTC CTG GAA GAC GGG GTA 478 Val Ala Arg Ala Leu Ala His Gly Val Arg Val Leu Glu Asp Gly Val 145 150 155 AAT TAC GCA ACA GGG AAT TTG CCT GGT TGC TCC TTT TCT ATC TTC TTG 526 Asn Tyr Ala Thr Gly Asn Leu Pro Gly Cys Ser Phe Ser Ile Phe Leu 160 165 170 175 CTT GCT CTT CTG TCA TGC ATC ACA GTG CCA GTG TCT GCA GTG GAA GTC 574 Leu Ala Leu Leu Ser Cys Ile Thr Val Pro Val Ser Ala Val Glu Val 180 185 190 AGG AAC ATC AGT TCT AGC TAC TAT GCC ACT AAT GAT TGC TCG AAC AGC 622 Arg Asn Ile Ser Ser Ser Tyr Tyr Ala Thr Asn Asp Cys Ser Asn Ser 195 200 205 AGC ATC ACC TGG CAG CTC ACC AAC GCA GTT CTC CAC CTC CCT GGA TGC 670 Ser Ile Thr Trp Gln Leu Thr Asn Ala Val Leu His Le u Pro Gly Cys 210 215 220 GTC CCA TGT GAG AAT GAC GAC GGC ACC CTG CAC TGT TGG ATA CAA GTA 718 Val Pro Cys Glu Asn Asp Asp Gly Thr Leu His Cys Trp Ile Gln Val 225 230 235 ACA CCT AAT GTG GCT GTG AAA CAC CGA GGC GCA CTC ACT CAC AAC TTG 766 Thr Pro Asn Val Ala Val Lys His Arg Gly Ala Leu Thr His Asn Leu 240 245 250 255 AGA ACG CAC GTT GAC ATG ATC GTC ATG GCA GCT ACG GTC TGC TCG GCC 814 Arg Thr His Val Asp Met Ile Val Met Ala Ala Thr Val Cys Ser Ala 260 265 270 TTG TAC GTG GGA GAC GTG TGC GGG GCC GTG ATG ATT GTG TCG CAA GCT 862 Leu Tyr Val Gly Asp Val Cys Gly Ala Val Met Ile Val Ser Gln Ala 275 280 285 CTC ATA ATA TCG CCA GAG CGC CAC AAC TTC ACC CAA GAG TGC AAC TGT 910 Leu Ile Ile Ser Pro Glu Arg His Asn Phe Thr Gln Glu Cys Asn Cys 290 295 300 TCC ATC TAT CAG GGC CAT ATC ACC GGA 937 Ser Ile Tyr Gln Gly His Ile Thr Gly 305 310

[Brief description of drawings]

FIG. 1 shows the HCVR6 strain and R by PCR.
The cloning strategy of 260 strain is shown. In the figure, the numbers on the upper side of each region indicate the position of the nucleotide sequence on HC-J6, and the numbers on the lower side indicate the name of the PCR primer used. The number in parentheses indicates the length (bp) of the DNA fragment.

FIG. 2 shows the steps for constructing a cloning vector pBM.

FIG. 3 is an electrophoretic (Western blot) photograph for confirming a positive reaction of non-A non-B hepatitis patient sera (lanes 1 to 5) with an HCV (R260) -derived gene expression product. Here, M is a molecular weight marker, and lanes 6 to 10 are sera of healthy persons.

Continuation of the front page (51) Int.Cl. ⁵ Identification number Office reference number FI Technical display part // (C12N 1/21 C12R 1:19) (C12P 21/02 C12R 1:19) (72) Inventor Nomoto Akio 3-18-22 Honkomagome, Bunkyo-ku, Tokyo Within the Tokyo Metropolitan Institute of Clinical Medicine (72) Inventor Michiho Ohara 3-18-22 Honkomagome, Bunkyo-ku, Tokyo Foundation Tokyo Metropolitan Institute of Clinical Medicine (72) Inventor Kyoko Ohara 3-18-22 Honkomagome, Bunkyo-ku, Tokyo Inside the Tokyo Metropolitan Institute of Clinical Medicine (72) Inventor Kiichi Sawai 35, Higashi-Tobori-cho, Higashi-ku, Nagoya, Aichi Prefecture Sanwa Co., Ltd. Inside the Chemical Research Laboratory (72) Inventor Masanori Kurono 35 Higashi Sotobori-cho, Higashi-ku, Nagoya, Aichi Stock Company Sanwa Chemical Research Institute (72) Inventor Takahiko Mitani 35 Tohigato-Hori-cho, Higashi-ku, Nagoya, Aichi Sanwa Chemical Co., Ltd. In-house (72) Inventor Yukiya Asano 35 Higashi Sotoboricho, Higashi-ku, Nagoya, Aichi Stock Association Inside the Sanwa Chemical Research Institute (72) Inventor Noboru Maki 1-3-1 Nishitsurugaoka, Oi-cho, Iruma-gun, Saitama Tonen Corporation Research Institute (72) Inventor Kazuhiro Higashi 1-chome, Muroya, Nishi-ku, Kobe, Hyogo Prefecture No. 2 International R & D Center Co., Ltd. R & D Center (72) Inventor Hiroyuki Mori 1-1-2 Muroya, Nishi-ku, Kobe-shi, Hyogo International Reagents Co., Ltd. R & D Center (72) Inventor Yosuke Ota 1 Muroya, Nishi-ku, Kobe-shi, Hyogo Prefecture 1-2, Kokusai Reagent Co., Ltd. R & D Center

Claims (15)

[Claims] 1. A nucleic acid fragment containing a nucleotide sequence encoding an antigen of a structural and non-structural region of non-A non-B hepatitis virus obtained by genetic engineering from plasma of a non-A non-B hepatitis patient. 2. A nucleic acid fragment containing a nucleotide sequence encoding a non-A non-B hepatitis virus antigen represented by all or part of the amino acid sequence shown in SEQ ID NO: 1. 3. The nucleic acid fragment according to claim 2, wherein the nucleotide sequence is all or part of the sequence of nucleotide numbers 1 to 1755 shown in SEQ ID NO: 1. 4. A nucleic acid fragment containing a nucleotide sequence encoding a non-A non-B hepatitis virus antigen represented by all or part of the amino acid sequence shown in SEQ ID NO: 2. 5. The nucleic acid fragment according to claim 4, wherein the nucleotide sequence is all or part of the sequence of nucleotide numbers 2 to 1450 shown in SEQ ID NO: 2. 6. A nucleic acid fragment comprising a nucleotide sequence encoding a non-A non-B hepatitis virus antigen represented by all or part of the amino acid sequence shown in SEQ ID NO: 3. 7. The nucleic acid fragment according to claim 6, wherein the nucleotide sequence is the whole or a part of the sequence of nucleotide numbers 3 to 2093 shown in SEQ ID NO: 3. 8. A nucleic acid fragment comprising a nucleotide sequence encoding a non-A non-B hepatitis virus antigen represented by all or part of the amino acid sequence shown in SEQ ID NO: 4. 9. The nucleic acid fragment according to claim 8, wherein the nucleotide sequence is all or a part of the sequence of nucleotide numbers 1 to 1386 shown in SEQ ID NO: 4. 10. A nucleic acid fragment comprising a nucleotide sequence encoding a non-A non-B hepatitis virus antigen represented by all or part of the amino acid sequence shown in SEQ ID NO: 5. 11. The nucleic acid fragment according to claim 10, wherein the nucleotide sequence is the whole or a part of the sequence of nucleotide numbers 2 to 937 shown in SEQ ID NO: 5. 12. An expression vector in which the nucleic acid fragment according to any one of claims 1 to 11 is introduced into a cloning site in a vector existing downstream of a promoter. 13. A host cell containing the expression vector according to claim 12. 14. A method for producing a recombinant non-A non-B hepatitis virus antigen (poly) peptide, which comprises expressing the nucleic acid fragment according to any one of claims 1 to 11 in a suitable host cell. A step of constructing a replicable expression vector to be obtained, a step of introducing the expression vector into a host cell to obtain a transformant, culturing the transformant under conditions capable of expressing the nucleic acid fragment, and carrying out the recombination A method comprising a step of expressing a (poly) peptide and a step of recovering the recombinant (poly) peptide. 15. A recombinant (poly) peptide obtainable by the method of claim 14.