JP3268502B2 - Non-A non-B hepatitis virus-related polynucleotide, polypeptide - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to an antigen related to non-A non-B hepatitis virus (hereinafter abbreviated as "NANB hepatitis virus"), an antibody, a detection system using the same, and NANB useful for their production. Hepatitis virus RNA, DNA
And polypeptides.

[0002]

2. Description of the Related Art Hepatitis caused by a virus has already been identified with the DNA and RNA of the virus itself. Hepatitis B, hepatitis A, D
Hepatitis B and E. However, for non-A non-B hepatitis (hereinafter referred to as "NANB hepatitis"), which is primarily a blood-borne infection and does not belong to the above classification, many researchers worldwide have searched for the causative virus. Nevertheless, the virus itself had not been elucidated.

In 1988, Chiron acquired NAN
It has been reported that an RNA virus genome named hepatitis C virus (hereinafter abbreviated as "HCV") as a causative virus of hepatitis B was successfully cloned, and its nucleotide sequence was announced. Based on this, an HCV antibody measurement system has been developed and is now being used for screening transfused blood and diagnosing patients. This HCV antibody measurement system is certainly partially related to NANB hepatitis. However, serious problems remain, such as the carrier and the capture rate of chronic hepatitis being only about 70%, and the inability to detect antibodies in the acute phase.
The response to hepatitis B remains unresolved. On the other hand, a method for detecting hepatitis B virus has been established, and in Japan, 95% or more of post-transfusion hepatitis is NANB.
Hepatitis is estimated to occur in about 280,000 cases annually.
The course of NANB hepatitis is poor, with most cases expected to become carriers and progress to chronic hepatitis. In addition, since the rate of progression to cirrhosis and liver cancer progresses at a very high rate over 10 to 20 years, it is desired to elucidate the body of the virus as soon as possible and to develop a diagnostic agent.

[0004]

SUMMARY OF THE INVENTION
Since there is a large number of NANB hepatitis that cannot be diagnosed with the V antibody detection kit, development of a diagnostic agent for NANB hepatitis with even better specificity is required. In order to advance this development, analysis at the gene level and amino acid level of NANB hepatitis virus is indispensable. The object of the present invention is
By elucidating the nucleotide sequence encoding the structural protein of NB hepatitis virus and analyzing it at the amino acid level based on this, an effective polypeptide was found as an antigen, and the detection system for NANB hepatitis virus, related antigens and antibodies was identified. Is to establish it.

[0005]

Means for Solving the Problems In order to find a polypeptide effective as an antigen, the present inventors have isolated RNA of NANB hepatitis virus from serum of human and chimpanzee carriers, and obtained a gene containing a non-coding region of the virus. The cDNA covering the entire region was cloned, its nucleotide sequence was determined, and the amino acid sequence was further examined. As a result, we found that the core protein, the shell protein in the structural gene of the virus, and the R protein in the nonstructural gene.
The present inventors have completed the present invention by finding specific regions encoding NA-dependent polymerases and studying them at the gene and amino acid levels.

The gist of the present invention is as follows. The NANB hepatitis virus RNA of the present invention was obtained as follows, and its nucleotide sequence was determined. Five specimens were obtained from human and chimpanzee plasma. HC-J1, HC-J5, HC-J
6 was obtained from a Japanese blood donor, and HC-J7 was obtained from a Japanese renal dialysis patient, and all were obtained from samples determined to be HCV antibody-positive. HC-J4 was derived from a sample obtained from a chimpanzee confirmed to be infectious for NANB hepatitis, but this was (-) for the HCV antibody from Chiron.
RNA was extracted from the five sample plasmas. Furthermore, each R
The nucleotide sequence homology between NAs was examined, and as shown in the Examples, about 1900 to 2500 nucleotide sequences at the 5 'end and about 1100 nucleotides at the 3' end of the extracted RNA were determined. Among them, the 5 'end of the three strains of HC-J1, J4 and J5 has already been partially disclosed in Japanese Patent Application Nos. 2-153401 and 2-304405, but this time newly added HC-J6. , J2, including two strains, the non-coding region of all strain genes, the region encoding the structural protein, and a part of the region encoding the non-structural protein,
The present invention has been completed.

As shown in Embodiment 1, HC-J1, HC
RNAs from strains J4, HC-J5, HC-J6 and HC-J7 are all 340 to 341 from the 5 'end.
5 'untranslated region consisting of 5 nucleotides, followed by a region encoding a structural protein (1149 bases)
Followed by the region encoding the nonstructural protein,
It has 1885 to 2551 nucleotides.
Based on this finding, the polynucleotides of SEQ ID NOs: 1 to 5 were obtained.

Further, as shown in Embodiment 3, HC-J
In 1, J4, J5, J6, and J7, the 3′-terminal side of RNA was found to have a non-coding region gene of 77 bases or more including T-stretch followed by 1096 bases encoding a nonstructural protein, Based on this, the polynucleotides of SEQ ID NOs: 6 to 10 were obtained.

[0009] It should be noted that EP-A-388232.
No. (Chiron), the nucleotide sequence of the present invention is HC-J1, J4, J4 with respect to the 2577 bases at the 5 'end including the non-coding region.
6, 95.2%, 80.5%,
It has 72.5% and 71.6% homology, and the HC-J5 strain shows only 76.8% homology for 1468 bases. This suggests that the HC-J1 strain is relatively closely related to the virus strain identified by Chiron and is an American virus, whereas the HC-J4, J5, J6, and J7 strains are slightly different. Type virus.

Based on the nucleotide sequences of the HC-J1, J4, J5, J6, and J7 strains, the amino acid sequence encoded by the NANB hepatitis virus 5'-terminal and 3'-terminal coding region genes was determined. As a result, the polypeptide encoded by each strain was HC-J1 (5 'end: 7
33, 3 'terminal side: 365), and J4 (7
33, 365), J5 (515, 365), J
6 (737, 365), J7 (719, 365)
Pieces). Based on the knowledge of the determined amino acid sequence, the polypeptides represented by SEQ ID NOS: 11 to 20 were obtained by chemical synthesis.

[0011] Two strains, HC-J1 and HC-J4, are 5 '
Comparing the terminal amino acid sequences, the homology of the amino acid sequence is higher in the upstream region than in the downstream region, and the upstream region contains a large amount of basic amino acids such as arginine and has high hydrophilicity. Basic amino acids are commonly found in core proteins and impart affinity to nucleic acids. Therefore, the upstream region encodes the viral core protein, the downstream region has many amino acid substitutions, and the eight sugar chain binding sites are common. Thus, it was inferred that the protein encoded an outer envelope protein.

When the homology of the 5'-terminal amino acid sequence among the strains is compared separately in the upstream region and the downstream region, 96.9 is found between HC-J1 and HC-J4 in aa1-191 presumed to be the core. %, 95.3 between HC-J6 and HC-J7
%, And 90.6% between HC-J1 and HC-J6, and 9% between HC-J4 and HC-J7.
It is slightly lower at 0.6%. In addition, in aa192 to 383 estimated to be the envelope portion, HC-J1 and HC-J
4 and 71.4% between HC-J6 and HC-J7, which are considerably lower than the core portion. Further, between HC-J1 and HC-J6, 60.9%, and between HC-J4 and HC-J7, 50.9%.
It showed a very low homology of 2.6%. The high homology of the amino acid sequence of the core region between the strains means that the antibody detection system using the polypeptide of the present invention can widely detect NANB hepatitis virus infection regardless of the American type or the Japanese type.

The present inventors selected the polypeptides of SEQ ID NOS: 21 to 27 from the study of the hydrophilicity score and synthesized them (claims 1 to 3). These polypeptides include HC-J1, HC-J4, HC-J
5, derived from HC-J6 and HC-J7 strains. Also,
Even if these have partial amino acid substitutions, they are effective as long as they do not lose antigenicity, and all are included in the present invention. The present inventors have synthesized the polypeptides of SEQ ID NOS: 21 to 27 and confirmed that the NANB hepatitis virus antibody measurement system using the polypeptides can detect NANB hepatitis virus with high specificity.

On the other hand, obtaining an envelope region protein is indispensable for vaccine development for the purpose of protecting the virus from infection, and the homology of the amino acid sequence can be roughly classified into the American type and the Japanese type. This means that not only the virus strain found by Chiron but also the amino acid sequence of the Japanese virus strain disclosed in the present invention is very important for vaccine production. HC
V, that is, hepatitis C virus is considered to be closely related to flavivirus based on its gene structure, and the 5 'non-structural partial base (nt
1491-) is presumed to correspond to a glycoprotein-encoding portion called NS1 in flavivirus.
NS1 is a type of flavivirus that is a yellow fever virus.
Have been reported to be involved in immune defense (Schlesinger et a).
l. , J. et al. Virol. , 60,1153,198
6), NA using the base and amino acid sequence of the present invention
A protein useful for treatment and prevention of NB hepatitis can be obtained. On the other hand, the homology between the strains at the 3 'terminal side is
87.4% of 365 amino acids between HC-J1 and HC-J4 and 89.9% between HC-J6 and HC-J7.
While it is 6%, it is slightly lower at 73.7% between HC-J1 and HC-J6. Also, the non-code area 39
The base was also 64.1% between HC-J1 and HC-J4, and 82.1% between HC-J6 and HC-J7.
% Compared to HC-J1 and HC-J6, which is considerably lower at 30.8%, reflecting the difference between the American type and the Japanese type, and the fact that there are many mutations among the Japanese types. Was assumed.

HCV, ie, hepatitis C virus, is considered to be closely related to flavivirus based on its gene structure, and the 3′-terminal nonstructural partial base disclosed in the present invention is called NS5 in HCV. , It is presumed to be a part encoding an RNA-dependent polymerase. Therefore, setting up an antibody detection system using the polypeptide encoded by NS5 is of great significance in diagnosing NANB hepatitis. Furthermore, elucidation of the nucleotide sequences of the NS5 region of the Japanese type and the American type in the present invention and obtaining the amino acid sequence of the polymerase are extremely useful as a means for treating and preventing NANB hepatitis virus and for researching them. As described above, in the present invention, gene regions encoding structural proteins and non-structural proteins that play an important role in the antigenicity of NANB hepatitis virus were identified, and their nucleotide sequences were clarified. Further, thereby, the amino acid sequences constituting the structural protein and the non-structural protein were clarified, and a material which was decisively useful for searching and synthesizing an antigenic polypeptide was provided.

Each of the polypeptides of the present invention can be obtained not only by chemical synthesis but also as an expressed peptide by known gene recombination. The present inventors also produced antibodies against each of the above polypeptides, and
It was confirmed that it can be used for detection of NB hepatitis virus antigen. Therefore, the detection system using each of the polypeptides of the present invention or their partial substitutes (claims 1 to 3), or an antibody against them is effective as a specific diagnostic agent for NANB hepatitis, Its practical value is great. All of these are included in the scope of the present invention.

Further, the above-mentioned polypeptides and antibodies thereto can also be used as raw materials for vaccines.

[0018]

The polypeptide of the present invention (Claims 1 to 3) or an antibody against the polypeptide is effective as a detection system for NANB hepatitis, a specific diagnostic agent, and the polynucleotide of the present invention (Claim 1). Paragraphs 4 to 6) can be incorporated into host cells to produce the peptide of the present invention.

[0019]

EXAMPLES Examples of the present invention will be described below, but the present invention is not limited to these examples. Example 1 The nucleotide sequence and the amino acid sequence of the 5 'end of NANB hepatitis virus were determined as follows.

(1) RNA extraction Specimens (HC-J1, J5, J6) obtained from plasma of Japanese blood donors and determined to be HCV antibody-positive (by an ortho HCVAb ELISA test of Ortho Diagnostic Systems Co., Ltd.) ), A sample obtained from a Japanese renal dialysis patient and also determined to be HCV antibody-positive (HC-J
7) RNA was extracted from HCV antibody (-) specimens (HC-J4) obtained from chimpanzees whose infectivity of NANB hepatitis was confirmed as follows. 1.8
1 ml of Tris-HCl buffer (10 mM, p
H8.0) and centrifuged at 68 × 10 ³ rpm for 1 hour. 200 mM NaCl, 10 m
M EDTA, Tris-HCl buffer (50 mM, pH 8.0) containing 2% (w / v) sodium dodecyl sulfate (SDS) and 1 mg / ml proteinase K were added, and the mixture was added at 60 ° C.
After heating for 1 hour and extracting with phenol / chloroform, ethanol precipitation was performed to obtain RNA.

(2) cDNA synthesis RNA extracted from HC-J1 plasma was heated at 70 ° C. for 1 minute, and 10 units of reverse transcriptase (c
DNA Synthesis SystemPlus,
Amersham Japan) and 20 pmol of oligonucleotide primer (20 mer) were added to obtain 42
The mixture was reacted at 1.5 ° C. for 1.5 hours to obtain cDNA. As a primer, # 8 (5 ′) was synthesized with reference to the nucleotide sequence of HCV shown in European Patent Application No. 8831022.5.
-GATGCTTGCGGAAGCAATCA-3 ')
Was used.

(3) Amplification of cDNA by polymerase chain reaction (PCR) was performed as follows.
DNA thermal cycler (Perkin-Elmer)
-Cetus) using the Gene Amp DNA amplification reagent kit (Perkin-Elmer-Cetus) and the method of Saiki et al. (Science 239, 48).
7-491.1988) with 35 cycles of cDN
A amplification was performed.

(4) Determination of J1, J4, 5 ′ terminal nucleotide sequence by construction of cDNA library HC-J1, HC-J4 using cDNA library
Fig. 1- (a) shows the nucleotide sequence analysis at the 5 'end of the genome.
As shown in (b), the cDNA was converted to bacteriophage λ.
Both the analysis of the clone obtained by inserting it into gt10 and the analysis result of the clone obtained by amplifying the cDNA by PCR were determined together. FIG. 1 shows the 5 'end of the NANB hepatitis virus genome along with restriction enzyme cleavage sites, and also shows the positions of the primers used. In the figure, the solid line indicates the range in which the base sequence was determined by using the clone of the bacteriophage λgt10 library, and the dotted line indicates the range in which the base sequence was determined by using the clone by PCR. Nt45 of HC-J1
The 1656 bases from 4 to 2109 were obtained by converting the cDNA obtained from the primer # 8 into a λgt10 phage vector (Amersh).
am), and was determined by clone φ41 obtained by insertion into am). Next, nt8 synthesized based on this sequence
24-843 new primer # 25 (5'-TCC
Using CTGTTGCATAGTTCACG-3 ′), four cDNA clones φ60, φ61, φ66, and φ75 were sequentially obtained from the HC-J4 cDNA library, and the base sequence of nt18 to 843 upstream was determined. HC
-For J4, antisense primer # 36 of nt 246-265 to identify the further 5 'end
(5'-AACACTACTCGGCTAGCAGT-
3 ′), cDNA is synthesized by terminal deoxynucleotidyltransferase.
A dA was added to the 5 'end of A, and two-step one-si
dedPCR amplification was performed, that is, the first time was performed using oligo dT primer (20-mer) and nt 188 to 207.
Antisense primer # 48 (5'-GTTGAT
35 cycles of PCR amplification using CCAAGAAAGGACCC-3 '), and the second time using the PCR product as a template, oligo dT primer (20-mer) and n
Antisense primer # 109 at t140 to 160
(21-mer; 5'-ACCGGATCCGCAGA
30 cycles of PCR using CCACTAT-3 ')
Was done. The obtained PCR product was subcloned into an M13 phage vector. Six independent clones, C896, believed to have the full-length 5 'terminal sequence.
2, C8968, C8970, C8974, C903
4. C9036 was obtained, and the nucleotide sequence of nt 1 to 17 was determined.

On the other hand, the sequence upstream of HC-J1 is
For nts 18 to 843, primer # 44 (5′-
GCGACACTCCACCCATGAAT-3 ') and #
25 (5'-TCCCTGTTGCATAGTTCAC
G-3 ′) clone C obtained by PCR using
2503, C2508, and C2510. Further upstream, the 5 'end was composed of 16 clones, C8931 and C8, obtained by the same method as HC-J4.
932, C8935, C8937, C8942, C89
44, C8949, C8950, C8951, C895
4, C8955, C9023, C9026, C903
Using 0, C9031, and C9032, the base sequence of nt1 to 37 was determined. Next, in order to determine the nucleotide sequence downstream of the 5 'end of HC-J1, primer # 148 (5'-
TGCACCTGGGATGAACTCAAC-3 ') and # 146 (5'-AGTAGCATCATCCACAA)
GCA-3 ') was used for 30 cycles of PCR. C11444, C11450 of the obtained clone,
The nucleotide sequence of nt 1984 to 2560 was determined by C11451. On the other hand, nt 738 to 1900 of HC-J4
The downstream sequence 1163 bases up to primer # 3
0 (5'-CTCATGGGGTACATTCCGCT
-3 ') and # 42 (5'-TCGGTCGTCCCCA
CCACAAC-3 '), three clones C2821, C3173, C3192 obtained by PCR.
Decided by To further determine the nucleotide sequence downstream of HC-J4, primer # 57 (5′-TATT
GCTCTCCCCCAAGCCC-3 ') and # 146
(5'-AGTAGCATCATCCACAAGCCA-
Using 3 ′), 30 cycles of PCR were performed. Obtained clones C11462, C11463, C11464
As a result, the nucleotide sequence of nt 1860-2560 was determined.
According to the above analysis results, HC-J1 and HC-J4 were 5 ′
It was determined that they had the nucleotide sequences shown in FIGS. 1 and 2 on the terminal side (SEQ ID NOS: 1 and 2).

(5) Determination of nucleotide sequence at the 5 'end of J5 by construction of cDNA library The nucleotide sequence at the 5' end of the HC-J5 genome is shown in FIG.
As shown in (C), it was determined from the results of analysis of clones obtained by amplifying cDNA by PCR. RNA was extracted from the HC-J5 sample in the same manner as described above, and its sequence was determined. nt 6 to 1868 were determined from each clone obtained by PCR using the following combinations of primers.

To further specify the 5 'end, three cDNA clones, C8995, C8997 and C8998, were obtained in the same manner as in Example 1- (4).
１６０160 base sequences were determined.

According to the above analysis results, HC-J5 is 5 ′
It was determined to have the base sequence shown in SEQ ID NO: 3 on the terminal side. In the 5 ′ non-coding region in HC-J5, 5 consecutive nts 6 to 10 in HC-J1
One of the cytosines is missing, for a total of 340
Was found to have a noncoding region base.

(6) Determination of nucleotide sequence at J6 and 5 'terminal by construction of cDNA library The nucleotide sequence at the 5' terminal of HC-J6 genome is shown in FIG.
As shown in (d), it was determined from the analysis results of clones obtained by amplifying cDNA by PCR. RNA was extracted from the HC-J6 sample in the same manner as described above, and its sequence was determined. nt24 to 2551 were determined from clones obtained by PCR using the following combinations of primers.

To further specify the 5 'end, 13 cDNA clones, C9577, C9579, C9581, and C958 were prepared in the same manner as in Example 1- (4).
4, C9587, C9590, C9591, C959
5, C9606, C9609, C9615, C961
6. C9619 was obtained, and the nucleotide sequence of nt 1 to 160 was determined.

According to the above analysis results, HC-J6 was 5 ′
It was determined to have the base sequence shown in SEQ ID NO: 4 at the end. In the 5 ′ non-coding region in HC-J6, 5 consecutive nts 6 to 10 in HC-J1
One of the cytosines is missing, for a total of 340
It was revealed that the base had non-coding region bases.

(7) Determination of base sequence at J7, 5 'end by construction of cDNA library The base sequence at the 5' end of HC-J7 genome is shown in FIG.
As shown in (e), the cDNA was amplified by PCR and determined from the results of analysis of clones obtained. RNA was extracted from the HC-J7 sample in the same manner as described above, and its sequence was determined. The nts 24 to 2498 were determined from each clone obtained by PCR using the following combinations of primers.

In order to further specify the 5 'end, eight cDNA clones were prepared in the same manner as in Example 1- (4).
C10513, C10515, C10521, C105
54, C10558, C10568, C11231, C
11232 was obtained, and the nucleotide sequence of nt 1 to 160 was determined.

From the above analysis results, it was determined that HC-J7 had the nucleotide sequence shown in SEQ ID NO: 5 at the 5 'end. HC-J1, J4, J disclosed by this embodiment
The 5'-terminal nucleotide sequences of 5, J6 and J7 are different from the HCV sequence described in the aforementioned European Patent Publication No. 388,232 and have been disclosed for the first time by the present invention.

(8) Determination of amino acid sequence 5 ′ of each genome of HC-J1, J4, J5, J6 and J7
Based on the terminal nucleotide sequence, 733 amino acids (J1, J4), 515 amino acids (J5), 737 are encoded by the structural gene region starting from the start codon ATG, excluding 340 to 341 bases in the 5 ′ untranslated region. Amino acid (J6) and 719 amino acid (J7) sequences were determined. Each sequence is HC-J1 (P-733: SEQ ID NO: 1
1), HC-J4 (P-733-2: SEQ ID NO: 12),
HC-J5 (P-515: SEQ ID NO: 13), HC-J6
(P-737: SEQ ID NO: 14), HC-J7 (P-71
9: as shown in SEQ ID NO: 15).

(9) Specification of core region When the homology of the amino acid sequence constituting the structural protein is calculated between the two strains, HC-J1 and HC-J4,
96.6% for 191 (core region), aa 192 to 38
3 (envelope region): 77.6%, aa1 to 38
3 as a whole have 87.2% homology. HC-J1,
In any of the HC-J4, HC-J5, HC-J6, and HC-J7 strains, the upstream aa1-191 (core region) has much higher homology as compared with the downstream aa192-383 (envelope region). , Indicating that the preservation between strains was high. Looking at the hydrophilicity of the amino acids aa to 191, three high hydrophilic portions were observed in the upstream regions aa 1 to 120. This region is rich in basic amino acids such as arginine (28/12 in J1).
(0, 23%) It is highly likely that it encodes the core protein of NANB hepatitis virus. Furthermore, the present inventors have found from the examination of the hydrophilicity score that the 36 amino acid sequence of aa39 to 74 (CP-9) forms an epitope of the core protein. (Claim 2). CP-9 is SEQ ID NO: 22 (CP-9-1: J1), SEQ ID NO: 23 (CP-
9-2: J4), SEQ ID NO: 24 (CP-9-3: J5,
J6, J7). The following CP-
9-1, 2, and 3 are collectively referred to as CP-9. The nucleotide sequence CN-9 of the gene encoding this amino acid sequence is represented by SEQ ID NO: 29 (CN-9-1: J1) and SEQ ID NO: 30 (CN-
9-2: J4), SEQ ID NO: 31 (CN-9-3: J5)
As shown in FIG. HC-J6, CN-9 of HC-J7
The partial base sequence is a partially substituted sequence of SEQ ID NO: 31 as long as the amino acid code does not change, and corresponds to nt 456 to 563 of SEQ ID NOS: 4 and 5. (Claim 5
Item) Hereafter, CN-9-1, 2, and 3 are collectively referred to as CN-9. In Examples and Experimental Examples, CN-9-1 represents CN-9-2 and CN-9-3 as representatives.
CP-9-1 may be referred to as CP
-9-2 and CP-9-3 may be represented as CP-9, but CN-9-2, CN-9-3, and CP-
The same action and effect were also confirmed in 9-2 and CP-9-3. The same applies to CN-5 and CP-5. C
P-9 was obtained by chemical synthesis, but also CN-
9, or nt4 of the nucleotide sequence of SEQ ID NOS: 1 to 5
The polynucleotides corresponding to 56-563 were
It could also be obtained by integrating and expressing in a host cell such as Richia coli. The ability to identify the epitope of the core protein of NANB hepatitis virus enables the synthesis of synthetic peptides, expressed peptides by genetic modification, polynucleotide synthesis, production of antibodies, production of diagnostic drugs for NANB hepatitis, and application to products such as NANB hepatitis vaccine. Was opened.

Example 2 Using the peptide of the amino acid sequence CP-9 shown in Example 1, a NANB hepatitis virus antibody measurement system was established as follows. (1) Production of Peptide Based on Merrifield's solid-phase method, the amino acid at the C-terminal of the peptide is first condensed to cross-linked polystyrene,
Next, t-butoxycarbonyl amino acids are sequentially condensed one by one toward the n-terminal, and the amino acid sequence is Arg-Arg-Gly-Pro-Arg-Leu-.
Gly-Val-Arg-Ala-Thr-Arg-L
ys-Thr-Ser-Glu-Arg-Ser-Gl
n-Pro-Arg-Gly-Arg-Arg-Gln
-Pro-Ile-Pro-Lys-Val-Arg-
An Arg-Pro-Glu-Gly-Arg peptide (CP-9-1, SEQ ID NO: 22) was synthesized (Claim 2). After the synthesis, the mixture was hydrolyzed under reduced pressure at 6 ° C., 110 ° C. for 24 hours to confirm the amino acid composition. Similarly, polypeptides CP-9-2 and CP-9-3 having the amino acid sequences of SEQ ID NOS: 23 and 24 were synthesized. Similarly to CP-9, a polypeptide CP-10 having the amino acid sequence of SEQ ID NO: 21 was synthesized (claim 1). Similarly to CP-9, a polypeptide CP- having the amino acid sequence of SEQ ID NOS: 25 to 27 was synthesized. 5-
1, CP-5-2 and CP-5-3 were synthesized. (Claim 3). Hereinafter, CP-5-1, 2, and 3 are collectively referred to as CP-
Five. The gene encoding the amino acid sequence of CP-10 has the sequence of SEQ ID NO: 28 (CN-10).
CP-10 is CN-10 or SEQ ID NOS: 1 to 5
Polynucleotides corresponding to nt 354 to nt 410 in the described nucleotide sequence can be obtained by incorporating them into host cells and expressing them. The nucleotide sequence CN-5 of the gene encoding the amino acid sequence of CP-5 is SEQ ID NO: 32 (CN-5-1: J
1), SEQ ID NO: 33 (CN-5-2: J4) SEQ ID NO: 3
4 (CN-5-3: J6). CP-5
Represents a polynucleotide corresponding to CN-5 or a polynucleotide corresponding to nt 642 to 701 of the nucleotide sequence of SEQ ID NOS: 1 to 5,
It was obtained by integrating and expressing in a host cell such as scherichia coli.

(2) NANB hepatitis virus antibody measurement system The measurement system was performed by a sandwich method using a polyvinyl microtiter plate. 50 μl of the peptide of this example having a concentration of 5 μg / ml was used as the solid phase of the plate.
Was injected into the wells and allowed to stand at room temperature overnight. afterwards,
The plate was washed 5 times with a physiological saline solution containing 0.05% Tween 20. As an overcoat, 30% (v / v) bovine serum, 0.05% Tween 20, 150 mM N
100 μl of aCl buffer (hereinafter referred to as “CS buffer”)
, And allowed to stand at room temperature for 30 minutes. As a primary reaction, 10 μl of the sample was added to 50 μl of CS buffer containing 30% bovine serum for each well, and the reaction was performed at room temperature.
Shake for hours. After the completion of the reaction, washing was performed 5 times in the same manner as described above. Secondary reaction is performed by using Horseradishp
One ng of eroxidase-labeled mouse anti-human IgG monoclonal antibody (Fab 'fragment: 22G, Institute of Specialty Immunology) was dissolved in 50 μl of bovine serum and reacted by shaking at room temperature for 1 hour. Washing was performed 5 times in the same manner. After reacting hydrogen peroxide as a substrate and 50 μl of a solution of O-phenylenediamine as a coloring agent at room temperature for 30 minutes, 4N sulfuric acid was added for 5 minutes.
The reaction was stopped by adding 0 μl, and the absorbance was measured at 492 nm. The criterion is that the ALT value is 34 Karmen un
It shows a normal value less than or equal to it and anti-HCV
Negative donor samples were measured and the average absorbance was 0.25
(0.05 + 0.25 = 0.30) was defined as the cutoff value.

Example 3 (1) Determination of 3'-terminal nucleotide sequence by constructing cDNA library The 3'-terminal nucleotide sequence of each genome of HC-J1, J4, J5, J6 and J7 is shown in FIG. As shown in ()), it was determined based on the analysis results of clones obtained by amplification by PCR. Nt 1 to 9 of HC-J1, J4, J5, J6, J7
For each of the 38 938 base sequences, each sample was ligated with primer # 80 (5'-GACACCCGCTGTTTTGAC
TC-3 ') and # 60 (5'-GTTCTTACT
GCCCAGTTTAA-3 ') was performed, and it was determined from the nucleotide sequence of each amplification product. The resulting clone is shown below. The 3'-terminal nucleotide sequence downstream of nt939 was determined according to the following method. That is, RNA was extracted from each sample according to the method described in Example 1, and poly (A) was added to the 3 ′ end of the RNA using poly (A) polymerase.
CDNA synthesis was performed using oligo (dt) ₂₀ as a primer, and the resulting cDNA was used as a template for PCR. In the PCR, as a first step, a 20-mer oligonucleotide specific to each strain cDNA was used as a sense primer, and oligo (dt) ₂₀ was used as an antisense primer.
The obtained cDNA was then used for the second step P
A specific sense primer corresponding to the downstream from the first step as CR was used as an oligo (d) as an antisense primer.
t) _Performed using ₂₀ . The T4 DNA polylase was allowed to act on the amplification product obtained by the two-step PCR to make both ends blunt, then the 5 'end was phosphorylated with T4 polynucleotide kinase, and the M13mp19 phage vector Hin was used.
The nucleotide sequence was determined by subcloning into the cII site.

For each strain, the two types of primers used for PCR and the resulting clones are shown below. HC-J1 # 100 (5'-AAGGCTGCCATATGTGGCAA-3 ') # 91 (5'-GCCATATGTGGCAAGTACCT-3') Clone: C9707, C9714, C9719, C9724, C9726, C9730, C9737, C9738, C9741 and C9742. C9936, C9943, C9945, C9949) HC-J4 # 61 (5'-TTGCGAGTCTGGGAGACATCG-3 ') # 78 (5'-TGTCCCGCGCTAAGCTACTGT-3') Clones: C8761, C8764, C8776, C8784, C8796, C8796, C8886 , C8812, C8819, C8825, C8849, C8851) HC-J5 # 97 (5'-AGTC GGGCGTCCCCTCATCT-3 ') # 90 (5'-GCCGTTTGCGGCCGATATCT-3') Clone: C10820, C10827, C10829, C10843, C10844, C10848, C10849, C10864, C10865, C10872) HC-JCCTCGCTGCT-GCT-CT -3 ') # 90 (5'-GCCGTTTGCGGCCGATATCT-3') Clone: C10311, C10313, C10314, C10320, C10322, C10323, C10326, C10328, C10330, C10333, C10334, C10336, C10337, C10345, C10346C1034 , C10349, C10350, C10357) HC- 7 # 123 (5'-CTTAGAGCGTGGAAGAGTGCG-3 ') # 124 (5'-GCCATCTGTGGCCGTTACCT-3') Clone: C10801, C10804, C10807, C10809, C10811, C10812, C10814, C10815, C10818 The above analysis , HC-J1, J4, J5, J
6, J7 have SEQ ID NOs: 6, 7, 8,
It was determined to have the nucleotide sequences shown in FIGS. HC-J1, J4, J5, J6 disclosed by this embodiment
Although the nucleotide sequence at the 3 'terminal side of J7 overlaps in nt 7938 to 8866 (929 bases) of the HCV sequence described in the above-mentioned European Patent Publication No. 388,232, the sequence of the present invention is clearly different from that of the present invention. For the first time.

(2) Determination of amino acid sequence 3 ′ of each genome of HC-J1, J4, J5, J6 and J7
Based on the base sequence at the terminal side, 396 encoded by a non-structural region of 1096 bases excluding 77 bases of the 3 ′ untranslated region
The sequence of 65 amino acids was determined. Each sequence is HC-J1
(P-365-1: SEQ ID NO: 16), HC-J4 (P-
365-2: SEQ ID NO: 17), HC-J5 (P-365
-3: SEQ ID NO: 18), HC-J6 (P-365-4:
SEQ ID NO: 19) and HC-J7 (P-365-5: SEQ ID NO: 20).

EXPERIMENTAL EXAMPLE CP-9 thought to form an epitope in the core region
(P39-74, 36mer), 5 types of peptides with different lengths (919-36, P27-3)
6, P19-27, P10-27, P10-18) and synthesized, and the absorption test was performed. The absorption test was performed on the sample and 1
30 μl of each of the five kinds of peptides adjusted to a concentration of 00 μl / ml were mixed in an equivalent amount and reacted at 37 ° C. for 2 hours.
It was used for measurement. The measuring method is as shown in Example 1. The results are as shown in Table 4.

[0042]

[Table 4]

As a result of the measurement of the anti-CP9-positive sample, it was confirmed from the absorption rates of the samples 1 and 2 that the whole of CP-9 was a peptide retaining antigenicity.

Experimental Example Using the NANB hepatitis virus antibody measurement system established in Example 2, NANB hepatitis after blood transfusion was compared with a conventional anti-HCV measurement kit for liver disease samples. As a result, it was confirmed that the detection system of the present invention was far superior to the conventional measurement kit in both the specificity and the detection in the acute phase.

(1) Post-transfusion NANB hepatitis In this example, 7 units of blood transfusion were performed twice, and a case of developing NANB hepatitis showing abnormal liver function about 2 months later was examined. In this case, the blood of the donor was measured retrospectively with anti-HCV and the kit of the present invention. As shown in Table 1, the absorbance was> 2.000, indicating that the donor blood was positive.

[0046]

[Table 1]

In the patient serum, two months after transfusion,
While the measurement system of the present invention shows a positive value exceeding the absorbance of 2 with an increase in liver function, anti-HCV shows (+) for the first time at the third month, one month behind the present invention. As described above, in the antibody measurement system of the present invention, a significant difference was observed in both the specificity of detection in the blood of the donor and the earlier detection of the antibody in the serum of the patient, compared to the anti-HCV measurement system. Was.

(2) Anti-CP9 in Liver Disease Samples The results of measuring the anti-CP9 positive rate for each liver disease sample classification are shown in Table 2.

[0049]

[Table 2]

Anti-CP9 was positive in 13 of 19 (68%) sporadic NANB acute hepatitis cases and 15 of 18 (83%) in post-transfusion NANB hepatitis cases. Among NANB chronic liver disease cases, 103 out of 133 cases (77%) of chronic hepatitis and 70 out of 113 cases of cirrhosis (62 cases)
%), And 31 out of 41 cases (76%) of liver cancer were positive. On the other hand, in acute hepatitis A, one out of eight cases was low positive, but in acute hepatitis B, all 11 cases were negative. 7% and 12% of chronic hepatitis B, cirrhosis and liver cancer, respectively.
% And 14% were positive. In autoimmune diseases, lupoid hepatitis and primary biliary cirrhosis, both cases were negative in 6 cases and 9 cases, respectively. Chiron's anti-
HCV data (Science 1989: 244:
362-366), acute post-transfusion hepatitis NANB 7
0.8% (17/24), sporadic NANB hepatitis 5
Compared to 6.6% (34 out of 58 cases),
It was found that anti-CP9 was higher and superior in terms of the capture rate in acute hepatitis. According to Japanese data, 2.4% (2 out of 82 cases) of acute hepatitis B were positive (ortho catalog, clinical research edition),
Anti-CP9 did not cross at all as described above, indicating that the specificity of anti-CP9 was high.

(3) RNA detection of specimens that match or do not match with the ortho-anti-HCV kit The proportion of agreement between the results of measurement of anti-CP9 and anti-HCV is 49% for NANB acute hepatitis (at onset) and NA
In the case of NB chronic liver disease, 66 to 67% of chronic hepatitis, cirrhosis and liver cancer were all present. anti-HCV negative and ant
As shown in Table 2, the presence of HCV RNA was confirmed in 10 cases excluding 1 case among 11 cases of NANB-type chronic liver diseases positive for i-CP9 (absorbance of 2 or more). From the above results, in the diagnosis of NANB-type chronic liver disease, anti-C
It was confirmed that P9 was excellent in specificity and sensitivity.

(4) Comparison of Antibody Detection in NANB Acute Hepatitis Specimens In three cases of transfusion and four sporadic cases of NANB hepatitis, onset of hepatitis (Month), 3 months after onset 6 months later with 3 points anti-CP9 and a
The anti-HCV was measured, and the seroconversion time of both antibodies was compared. Table 3 shows the results.

[0053]

[Table 3]

Anti-CP9 was already positive at the onset of hepatitis in both transfused case 4 and sporadic case 3, whereas anti-HCV was positive at the onset of hepatitis in one of seven cases (sporadic). 5 cases are 3 months, the remaining 1
Examples are positive only after 6 months, NA
In acute hepatitis NB, anti-CP9 is anti-
It was confirmed that an antibody appearing earlier can be specifically detected as compared with HCV (FIG. 3). From the above, the diagnosis of NANB-type acute hepatitis was made possible by anti-CP9 of the present invention without using the conventional exclusion diagnosis.

Experimental Example An example of the measurement of NANB hepatitis virus antibody using a peptide having the amino acid sequence CP-10 (P5-23, 19-mer) is shown below. The measuring method is as shown in Example 2. a
Although anti-CP10 and anti-CP9 show almost the same positive rate, anti-C10
P10 is a case where the anti-CP9 negative sample is also positive, and is considered to supplement the measurement of anti-CP9. Samples 1 to 5 contain anti-HCV
(Chiron) is negative, but anti-CP10 and anti-CP9 are positive and NAN by PCR
BRNA has been detected. From this, anti-C
It was confirmed that the P10 measurement system was superior in both sensitivity and specificity to the anti-HCV kit (Table 5).

[0056]

[Table 5]

Experimental Example Amino acid sequence CP-5 (P101-120, 20 mer)
An example of the measurement of NANB hepatitis virus antibody using the above peptide is shown below. The measuring method is as shown in Example 2. When comparing anti-CP5 and anti-CP9, the positive rate of the same sample is higher in anti-CP9, but anti-CP5 is also observed in anti-CP9 negative sample as seen in three samples 8 to 10. It may be positive, and is considered to supplement the measurement system of anti-CP9. Samples 5 to 7 and sample 10 were anti-H
Although CV (Chiron) was negative, anti-CP5 and anti-CP9 were positive, and N-
ANB RNA has been detected. From this, an
The ti-CP5 measurement system is based on the anti-HCV kit.
It was confirmed that both sensitivity and specificity were excellent (Table 6).

[0058]

[Table 6]

[0059]

According to the present invention, a NANB hepatitis detection system which is capable of detecting NANB hepatitis virus which could not be captured by the conventional measurement method, has excellent specificity and sensitivity, and is capable of early detection is provided. provide. As a result, the patient can be diagnosed early and accurately, and the blood of the NANB hepatitis virus carrier can be removed at a high rate even in the screening of donor blood. In addition, the polyheptide of the present invention and an antibody against the same can be used for production of vaccines and immunological preparations, and the structural gene of NANB hepatitis virus provides essential information for finding polypeptide antigens and antibodies.

[Brief description of the drawings]

FIG. 1 shows a method for determining the nucleotide sequence of NANB hepatitis virus. (A) HC-J1 strain, (b) HC-J4
(C) HC-J5 strain, (d) HC-J6 strain,
(E) shows the method for determining the base sequence on the 5′-terminal side of the HC-J7 strain. (F) is a diagram showing a method for determining the 3 ′ terminal nucleotide sequence of each genome. In the figure, the solid line indicates the range in which the base sequence was determined by using the clone of the bacteriophage λgt10 library, the dotted line indicates the range in which the base sequence was determined by using the clone by PCR, and the clone name is shown beside the line.

FIG. 2 is a view showing hydrophilicity of amino acids of a structural protein of NANB hepatitis virus;

FIG. 3 is a graph showing changes in GOT value (filled black), -CP9 (open circle), and -HCV (filled circle) of hepatitis NANB patients after blood transfusion.

[Sequence list]

──────────────────────────────────────────────────の Continuation of the front page (51) Int.Cl. ⁷ identification code FI G01N 33/577 C12N 15/00 C (58) Field surveyed (Int.Cl. ⁷ , DB name) C12N 15/09 C07K 14 / 16 C12N 15/02 BIOSIS (DIALOG) GenBank / EMBL / DDBJ Medline (STN)

Claims (6)

(57) [Claims] 1. A polypeptide CP-10 having the amino acid sequence of SEQ ID NO: 21. 2. A polypeptide CP-9-1 having the amino acid sequence of SEQ ID NO: 22. 3. A polypeptide CP-5-1 having the amino acid sequence of SEQ ID NO: 25. 4. A polynucleotide CN-10 having the nucleotide sequence of SEQ ID NO: 28. 5. A polynucleotide CN-9-1 having the nucleotide sequence of SEQ ID NO: 29. 6. A polynucleotide CN-5-1 having the nucleotide sequence of SEQ ID NO: 32.