JPH0656896A - Fused protein having antigen protein originated from blood transmission type non-a, non-b hepatitis virus - Google Patents

Abstract

(57) [Summary] [Objective] To develop a fusion protein useful as a diagnostic agent for non-A non-B hepatitis virus infection. [Structure] A carrier in which a peptide (A) containing at least an epitope of a core protein derived from blood-borne non-A non-B hepatitis virus and a peptide (B) containing at least an epitope of another protein have isoelectric points in the acidic region. It is connected through protein (C) and has an isoelectric point of 6-1
0, a fusion protein having a molecular weight of 25-100 kilodaltons.

Description

Detailed Description of the Invention

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a novel protein useful as a diagnostic agent, and more specifically, at least a part of a core protein derived from blood-transmitted non-A non-B hepatitis virus and other antigenic proteins are acidic regions. The present invention relates to a fusion protein obtained by binding via a carrier protein having an isoelectric point.

[0002]

Viral hepatitis is a liver disease caused by infection with hepatitis virus. So far, hepatitis viruses of type A, type B, and type D (delta) have been separately identified as hepatitis viruses. However, it has been reported that there are many viral hepatitiss that are not caused by infection with these hepatitis viruses. Such hepatitis has been generically called non-A non-B hepatitis. Therefore, the pathogenic virus of non-A non-B hepatitis is not always one type, but actually, an infectious virus (transmitted through food and water) and a blood-borne virus (mainly transmitted through blood and after transfusion) It is reported that the existence of The non-A non-B hepatitis virus was discovered by cloning the non-structural protein region of the viral gene, unlike the history of the discovery of other viruses in the past (Science, 244 (4909) 359).
-362 (1989)).

This non-A non-B hepatitis virus is currently considered to be an enveloped virus. A C100-3 polypeptide in which a part of the nonstructural protein region of the viral genome is expressed in yeast as a fusion protein with human superoxide dismutase (SOD) is provided as a diagnostic agent, and an antibody assay system using the same is provided. (Ortho HCV ELISA Test
System) is established (Science, 2
44 (4909) 362-364 (1989)). However, an antibody against this non-structural protein is not produced immediately after virus infection, and thus there is a problem that it is difficult to detect it in the early stage of infection.

[0004] On the other hand, the gene for the structural protein region of blood-borne non-A non-B hepatitis virus was also cloned by several research groups (for example, W
O90 / 11089, WO90 / 14436, Okamoto et al .:
Japan J. Exp. Med. , 60 (3) 167
-177 (1990), Kato et al .: Jpn. J. Canc
er Res. , 81, 1092-1094 (199
0), Kato et al .: Proc. Natl. Acad. Sc
i. USA, 87, 9524-9528 (1990),
And Takamizawa et al .: J. Virol. , 65 (3) 119
5-1213 (1991)). Unlike non-structural proteins, antibodies against structural proteins are produced in the early stage of infection, so it is preferable to use structural proteins as diagnostic agents.

Further, based on comparison of nucleic acid sequences of blood-transmitted non-A non-B hepatitis virus and flavivirus, a core protein, which is considered to be one of the structural proteins, was used in insect cells in Escherichia coli or in a baculovirus vector. , And has been successfully expressed in animal cells (Muraiso et al .: Biochem. Biophys. Res. Com.
mun. , 172 (2) 511-516 (1990),
Matsuura et al .: The 49th Annual Meeting of the Japanese Cancer Society, p113, 19
1990, Matsuura et al .: The 38th Annual Meeting of the Japanese Society of Virology, Abstracts p. 47, 1990, Harada et al .: J. Virol. ,
65 (6) 3015-3021, (1991)).

The detection rate of the core antibody using the core protein is the Ortho HCV ELISA Test System.
It has been evaluated that it is superior to the detection rate by m 2 (Chiba et al .: Proc. Natl. Acad. Sc.
i. USA, 88, 4641-4645, (199
1)). Glutathione-S is a part of this core protein
-It has been reported that the epitope was found by binding to transferase (Proc. Natl. Aca
d. Soc. USA, 88, 5462- (1991)).
In addition, a peptide in the hydrophilic region of the core protein was chemically synthesized (Okamoto et al .: Japan J. Exp. Med.,
60 (4) 223-233, (1990)), non-A non-B
Attempts have been made to produce an assay system for detecting anti-core antibody in the serum of hepatitis C patients.

Generally, an ELISA is used as a diagnostic system.
(Enzyme-linked immunoadso
The first method has high sensitivity, but the operation is complicated, and thus is not suitable for processing a large amount of specimens. On the other hand, the latter is suitable for processing a large amount of sample.

However, since the above-mentioned core protein has a high isoelectric point and is basic, if it is carried on a support as it is, non-specific aggregation may occur depending on the support and it cannot be used as a diagnostic agent. There is. Further, if the synthetic peptide of the antigenic site is directly supported on the carrier, the molecular weight is too small and the sensitivity cannot be improved. Furthermore, as a diagnostic agent, it is generally important to prevent false negatives in an antibody test for blood-transmitted non-A non-B hepatitis virus for transfusion, and for that purpose, use an antigen having as many antigenic sites as possible. It was desired to do.

[0009]

Under these circumstances, the present inventors have found that the core protein derived from blood-transmitted non-A non-B hepatitis virus and other antigen proteins have acidic isoelectric points. We have succeeded in producing a fusion protein linked via a carrier protein, and the fusion protein has an isoelectric point, molecular weight, and
They found that they have antigenicity and completed the present invention.

[0010]

Thus, according to the present invention, a peptide containing at least an epitope of a core protein derived from a blood-transmitted non-A non-B hepatitis virus (hereinafter, simply referred to as non-A non-B hepatitis virus) ( A carrier protein in which the peptide (B) containing at least an epitope of another antigen protein has an isoelectric point in the acidic region (hereinafter referred to as an acidic carrier protein) (C)
A fusion protein having an isoelectric point (pI) of 6 to 10, preferably 6.5 to 9, and a molecular weight of 25 to 100 kilodalton (hereinafter referred to as Kd), preferably 28 to 40 Kd is provided. It

The peptide (A) containing at least an epitope of the core protein of the present invention is substantially free of the hydrophobic region of the core protein. The core protein which does not substantially include the hydrophobic region in the present invention means that the hydrophobic region of the core protein has about 10 amino acids or less,
It is preferably 5 or less, more preferably not contained at all, and has a molecular weight of 1 to 21 Kd, preferably 8
Specific examples of such a peptide include those having the amino acid sequence of the 1st to 169th amino acids described in SEQ ID NO: 1, CP9 protein and CP10 protein (Hepatology, 15, 2, 180-
186 (1992)) and the like. The hydrophobic region of the core protein is a region called a membrane region existing on the carboxy-terminal side of the core protein, and specific examples include those having the 170th to 190th amino acid sequences of SEQ ID NO: 4. Can be mentioned.

The peptide (B) containing at least an epitope of an antigen protein other than the core protein is not particularly limited as long as it is a peptide containing an epitope of a blood-transmitting non-A non-B hepatitis virus. Envelope protein (J. Viro
l. , 65, 3, 1150-1113 (1991)) and NS4 protein (J. Vir) which is a non-structural protein.
ol. , 65, 3, 1150-1113 (199
1)), NS3 protein (J. Virol., 65,
3, 1150-1113 (1991)). A specific example of such a protein is a peptide containing an epitope of NS4 protein shown in SEQ ID NO: 2.

The acidic carrier protein (C) is not an antigen protein derived from human pathogenic bacteria and viruses and has an isoelectric point (pI) of acidic,
Preferably pI4 to 6.5, more preferably pI5 to 6
What is necessary is. Specific examples of such an acidic carrier protein include glutathione-S-transferase derived from mouse parasite (EC2.5.1.
18; hereinafter referred to as GST), cytochrome C oxidase (EC1.9.3.1), and the like, but GST is preferable. By combining this acidic carrier protein with the core protein, it is possible not only to lower the isoelectric point of the fusion protein, but also to allow the acidic carrier protein to exist between the epitope of the core protein and the epitope of other proteins. It is possible to prevent the epitopes of the core protein and other antigenic proteins from being too close to each other in terms of a three-dimensional structure, and sufficiently exert their antigenicity.

Specific examples of the fusion protein of the present invention include those having the amino acid sequence shown in SEQ ID NO: 3 consisting of a part of core protein, glutathione-S-transferase and a part of NS4 protein, and of the core protein. Some of them are composed of part of glutathione-S-transferase and part of the envelope protein, some of core proteins are composed of part of cytochrome-C-oxidase and part of NS3 protein, and the like.

The fusion protein of the present invention is usually produced by expressing in a host cell a recombinant vector in which a gene encoding the fusion protein is incorporated into an appropriate vector.

The gene encoding an antigen protein derived from non-A non-B hepatitis virus has, for example, a GPT of 100 or more, and viral RNA is extracted from blood of a Japanese person diagnosed with non-A non-B hepatitis by known techniques. After isolation and cDNA preparation with reverse transcriptase, a PCR reaction using primers designed based on the known sequence of non-A non-B hepatitis virus (see Takamizawa et al., Supra) (Saiki et al .:
Magazine Science, 239, 487-, (198
8)) can be inserted into a cloning vector and cloned.

The gene encoding the fusion protein is ligated based on the fusion protein designed in advance according to a conventional method using an appropriate synthetic adapter so that the reading frame of the gene encoding each protein is not displaced. And a specific example of such a gene having the base sequence shown in SEQ ID NO: 3.

As a vector used for producing a recombinant vector, a vector used in a general gene recombination technique may be used. For example, pBR322 and pBR3.
25, plasmids such as pUC18, λ-phage,
Phage such as M13 phage, pHC79 (Gen
e, 11, 291 (1980)) and the like.

A method for producing a recombinant vector by incorporating a gene encoding a fusion protein into a vector may be carried out by a conventional method. For example, after cleaving the vector with a restriction enzyme, the above gene may be prepared under the control of an appropriate expression control sequence. Should be directly connected. The expression control sequence used may be one that functions in E. coli, and examples thereof include a lac promoter / operator, trp promoter, tac promoter, lpp promoter, and PL promoter.

A recombinant vector incorporating a gene encoding a fusion protein can be transformed into Escherichia coli to obtain a microorganism capable of producing the fusion protein.

Whether or not the desired fusion protein is expressed can be analyzed by a conventional method. For example, the transformant is cultured under inducing conditions, and the resulting host cell is disrupted. After recovering the crude protein, SD
After fractionation by S-polyacrylamide gel electrophoresis, the gel can be confirmed by staining with Coomassie Brilliant Blue stain or by immunoassay using serum of non-A non-B hepatitis patients.

The fusion protein is purified by appropriately combining well-known conventional methods such as salting out, gel filtration, ion exchange and separation by affinity column chromatography, high performance liquid chromatography, and fractionation by electrophoresis. I can. The molecular weight and isoelectric point of the purified fusion protein can be measured by a conventional method. For example, the molecular weight may be measured by SDS polyacrylamide gel electrophoresis, and the isoelectric point may be measured by gel electrophoresis using Ampholine (Pharmacia LKD Biotechnology; pH range 3.5-9.8). .

The fusion protein of the present invention thus obtained has an isoelectric point of 6 to 10, preferably 6.5 to 9,
Molecular weight is 25 to 100 Kd, preferably 28 to 40 Kd
belongs to.

Such a fusion protein can be used as a diagnostic agent for non-A non-B hepatitis virus by supporting it on a support according to a conventional method. The support used for producing the diagnostic agent is not particularly limited as long as it is used for the diagnostic agent, and examples thereof include microplates, polystyrene beads, latex particles, and membranes.

The method for supporting the fusion protein on the support may be any method suitable for the support.
As a method for adsorbing to a microplate, there is a method in which the fusion protein is dissolved in an appropriate buffer solution, added to the plate and then left at room temperature. The diagnostic agent produced in this manner is a commonly used assay method for antigen-antibody reaction, such as RI.
A (radio immunoassay), ELISA
Method, fluorescent antibody method, passive agglutination reaction, reverse passive agglutination reaction and the like.

[0026]

According to the present invention, therefore, a core protein derived from blood-transmitting non-A non-B hepatitis virus having an isoelectric point and a molecular weight suitable for a diagnostic system by the agglutination method and containing a plurality of antigen sites. A fusion protein consisting essentially of and other antigenic proteins and an acidic carrier protein is obtained. This fusion protein can be used for diagnosis of blood-transmitted non-A non-B hepatitis virus infection.

[0027]

EXAMPLES The present invention will be described in more detail with reference to the following examples. Example 1 Recombinant plasmid pGEX-CV3654
(A) Gene cloning of blood-borne non-A non-B hepatitis virus structural protein region 0.5 m of human serum negative for hepatitis B and having a GPT value of 100 or more
5 times volume of guanidinium thiocyanate solution (4M guanidinium thiocyanate, 50 mM Tris-HCl pH
7.6, 10 mM EDTA, 0.1 M 2-mercaptomethanol, 2% sarcosyl) was added, phenol / chloroform extraction was performed, and total RNA in the serum was purified by ethanol precipitation using glycogen as a carrier. on the other hand,
Based on the results of Okamoto et al. (See above), it has the following sequences4
Book synthetic DNA primers were made.

(1) 5'CATATGAGCACGAATCCTAA, (2)
5 'CCCAACGAGCAAGTCGACGT, (3) 5' TGATCATGCATACTCC
CGGGT, (4) 5'GCTGCCGTTGGTATTCACAA

Using the synthetic DNA of (4) from the above RNA as a primer, a cDNA was prepared using a cDNA synthesis system (Boehringer Mannheim). Synthesized cd
PCR was performed using primers (1) and (2) with NA (about 1.26 kbp) as a template to obtain this cD.
The NA fragment was amplified. The 5'end of the amplified cDNA fragment was phosphorylated with T4 polynucleotide kinase and ligated with SmaI-digested pUC18, and the resulting plasmid of about 3.41 kbp was named pIKC. Further, the same operation as above was performed using the primers (3) and (4), and the obtained plasmid of about 3.45 kbp was p-typed.
It was named IKE. Then, the obtained pIKC and pIK
E was digested with the restriction enzyme AvaI and then ligated, and the resulting plasmid of about 4.01 kbp was named pIK4CE.

(B) Determination of nucleic acid base sequence and amino acid sequence of pIK4CE In order to examine the cDNA base sequence of the non-A non-B hepatitis virus cloned in pIK4CE prepared in (A) above, it was incorporated into M13 phage. 26 kbp c
The DNA fragment was recloned and the Sequenase Sequencing Kit (United States Bio)
The base sequence was determined by the chemical company). The nucleotide sequence obtained as a result and the decoding result of the amino acid sequence predicted therefrom are shown in SEQ ID NO: 4.

(C) Synthesis of DNA Encoding Amino Acid Sequence of Nonstructural Protein Protein C100- in Japanese Patent Publication No. 2-500880
3 (part of NS3 protein and NS4 protein) based on the amino acid sequence of 40 amino acids shown in SEQ ID NO: 2 (this 40 amino acids have an antigenicity
A DNA encoding a synthetic peptide consisting of amino acids was confirmed by reacting with serum of a patient identified as a blood-transmitted non-A non-B hepatitis patient) was prepared by chemical synthesis. That is, the base sequence shown in SEQ ID NO: 2 was designed in consideration of a suitable codon usage of E. coli as a DNA sequence encoding 40 amino acids, and 12 DNA fragments shown below were synthesized.

U1: 5'-GATCCGTGGTGCTGTCCGGC-3 ', L
1: 3'-GCACCACGACAGGCCGTTCGGCC-5 'U2: 5'-AAGCCGGCGATCATTCCGGA-3', L2: 3'-GCTAGTA
AGGCCTGGCGCTT-5 'U3: 5'-CCGCGAAGTCCTGTACCGCG-3', L3: 3'-CAGGACA
TGGCGCTTAA-5 ', U4: 5'-AATTCGATGAGATGGAAGAGTG-3', L4: 3'-GCTAC
TCTACCTTCTCACGAGAGTCGT-5 ', U55'-CTCTCAGCACTTACCGTACATCGAGCAA-3', L5: 3'-G
AATGGCATGTAGCTCGTTCCGTACTA-5 ', U6: 5'-GGCATGATGCTGGCCAATTAAGAATT-3', L6: 3'-C
GACCGGTTAATTCTTAAGATC-5 '

U1 and LI, U2 and L2, U3 and L3, U
Annealed U1 / L1, U2 / L2, U3 by annealing 4 and L4, U5 and L5, U6 and L6, respectively.
/ L3 is mixed and the restriction enzyme BamHI and the restriction enzyme Eco are mixed.
It was inserted into pGEX-2T (manufactured by Pharmacia) cut with RI using a DNA ligase. The resulting plasmid was named pGEX-CV1236. Also, pGE, which was also mixed with U4 / L4, U5 / L5, and U6 / L6, and cleaved with restriction enzymes EcoRI and XbaI
It was inserted into X-2T using DNA ligase. The resulting plasmid was named pUC-CV1254.

The 71 bp fragment recovered by cleaving the plasmid pUC-CV1254 with the restriction enzyme EcoRI was inserted into the EcoRI site of the previously obtained pGEX-CV1236 using a DNA ligase. The resulting plasmid was named pGEX-CV3654. This plasmid pGEX-CV3654 has a part of non-structural protein gene derived from non-A non-B hepatitis virus.

Example 2 Production of Fusion Protein C83Z3654 (A) Production of Fusion Protein Expression Vector The plasmid pIK4CE produced in Example 1 (A) was cleaved with restriction enzymes NdeI and FspI, and then blunt-ended by Klenow treatment, A DNA fragment of about 0.6 kbp was recovered from the agarose gel. The plasmid pKK223-3 (manufactured by Pharmacia) having the tac promoter was cleaved with the restriction enzyme EcoRI, blunt-ended by Klenow treatment, and the previously recovered 0.6 kbp DNA fragment was DNA.
Inserted using ligase.

This plasmid was introduced into E. coli TG1,
Inserted DNA from an ampicillin-resistant transformant that appeared
In the positive direction (direction of SEQ ID NO: 3 and ta of pKK223-3)
A colony having a plasmid inserted in the same direction as the c promoter transcription direction was selected). Forward plasmid (this is pKKFsp
(Named as) is selected with the restriction enzyme KpnI and the restriction enzyme Hind.
When cleaved with III, an approximately 0.34 kbp fragment appears, which allows easy selection.

Restriction enzyme K of this plasmid pKKFsp
Synthetic DNA adapter (5'-CCTATGGCCAT at the pnI site
AGGGTAC-3 ') was inserted into pKKF
It was named spAD. After cleaving pGEX-2T (Pharmacia) with the restriction enzymes BalI and PvuI,
A DNA fragment of about 1.4 Kbp was recovered. This DNA fragment contains the latter half of the glutathione-S-transferase gene. Then, pKKFspAD was cleaved with restriction enzymes BalI and PvuI, and then about 2 kbp
The DNA fragment containing the DNA replication initiation region was recovered.

The approximately 1.4 Kbp DNA fragment previously collected was ligated to this approximately 2 kbp DNA fragment, and the resulting plasmid was named pHC83Z. This plasmid pH
C83Z has a gene encoding a part of core protein and a gene encoding a part of GST.

Furthermore, pHC83Z is a restriction enzyme BamH.
After digestion with I and the restriction enzyme PvuI, the plasmid pGEX-CV3654 prepared in Example 1 was similarly digested with the restriction enzyme Ba.
The approximately 1.1 kbp DNA fragment recovered after digestion with mHI and PvuI was inserted, and the resulting plasmid was transformed into pHC8.
It was named 3Z3654. This plasmid has a gene encoding an epitope of core protein, a gene encoding GST, and a gene encoding an epitope of NS4 protein. The proteins encoded by these genes have epitopes (Proc. Nat. Acad. Sci. USA, 8
8, 5462-5466 (1991), JP-A-4-14
9298). This plasmid pHC83Z365
4 was introduced into Escherichia coli JM109, and the resulting transformed Escherichia coli was named JM109 / [pHC83Z3654].

(B) Expression of fusion protein C83Z3654 The transformed Escherichia coli JM109 / [p obtained in (A) above.
HC83Z3654] in 1 ml of LB medium supplemented with 50 μg / ml of ampicillin, and added to 1 mM isopropyl-β-D-thiogalactopyranoside (hereinafter, IPTG).
), And further cultured with shaking at 37 ° C. for 4 hours. The cells were collected by centrifugation, suspended in a Laemmli buffer, and boiled at 100 ° C. for 10 minutes to be solubilized. The lysate was analyzed by 15% SDS-polyacrylamide gel electrophoresis. Example 3 as control
Escherichia coli JM109 transformed with pHC83Z prepared in (A), transformed E. coli JM109 / [pHC83
Z] was treated similarly.

When the gel after electrophoresis was stained with Coomassie Brilliant Blue staining solution and then destained, a protein having a molecular weight of 33 Kd was observed as a major band in the lysate of transformed Escherichia coli JM109 / [pHC83Z3654]. This protein was not found in E. coli JM109 / [pHC83Z] transformed with pHC83Z. In addition, it was confirmed by immunoblotting that this protein specifically reacts with the serum of non-A non-B chronic hepatitis patients. This 33 Kd protein is C83Z36
It was named 54.

(C) Purification of expressed fusion protein C83Z3654 8 l LB medium supplemented with 50 μg / ml ampicillin
To the transformed Escherichia coli JM109 [p] obtained in (B) above.
HC83Z3654] has a turbidity of 0.15 at 660 nm.
Was inoculated so that This was shake-cultured at 37 ° C for 2 hours, then induced with 1 mM IPTG, further shake-cultured at 37 ° C for 4 hours, and the culture solution was centrifuged at 5,000 x G for 30 minutes to obtain 31 g of wet bacterial cells. It was This is 60 ml of PB
After suspending in S (10 mM potassium phosphate buffer, 0.85% sodium chloride (pH 7.5)), it was crushed with a French press (Otake Seisakusho Model 5615, 1500 kgf / cm 2) and 20 minutes at 15,000 × g. The precipitate was collected by centrifugation.

Then 60 ml of solubilization buffer (7 M urea, 20 mM dithiothreitol, 1% Triton X-1).
00, 50 mM Tris-HCl (pH 8.0)),
Ultrasonic crusher (Branson, sonifier I
After dispersion in I), the mixture was shaken overnight at room temperature to obtain a crude protein solution as a soluble fraction. This is a column of CM-Toyopearl (650M, manufactured by Tosoh Corporation) (diameter 2 cm × height 16).
cm), 0.2-0.8M in the presence of 6M urea
Elution was performed with a salt concentration gradient (400 ml),
The target fusion protein was eluted at a concentration of M sodium chloride. Thus, the fusion protein C83Z having a purity of 99% or more
About 10 mg of 3654 was obtained. The amino acid sequence of the obtained fusion protein is shown in SEQ ID NO: 3.

Example 3 (A) Measurement of Isoelectric Point of Fusion Protein Precast gel (Resofine FS-IEF, pH 3.5-9.5 and pH 9) manufactured by ACI Japan Co., Ltd.
-11) was set in the electrophoresis apparatus manufactured by the same company (F-CELLS and Handycool 8), and the fusion protein C83Z3654 (4 μg) purified in Example 3 (C) was placed on the gel sample well and cooled sufficiently. After that, electrophoresis was performed at a constant power of 3 W for 35 minutes. After the migration, fixation, staining and decolorization were performed. Calibration kit for isoelectric point made by Pharmacia (Product code No. 17
-0471-01) was run at the same time. As a result, the measured isoelectric point of the fusion protein C83Z3654 was 6.7.
Met.

(B) Examination of specificity of fusion protein for non-A non-B hepatitis The fusion protein C83Z36 obtained in Example 3 (C)
54 in E buffer (Na2CO3 1.6 g / l, NaHCO3 2.9 g / ml) so that the concentration becomes 0.1 μg / ml.
l, NaN3 0.16 g / l), and then 100
μl was added to an EIA plate (manufactured by Nunc) and left at room temperature for 1 hour. Discard the antigen protein solution and
% Tween20-containing phosphate buffer (hereinafter PBS-T
After washing twice with (w20), 200 μl of PBS-Tw20 containing 3% bovine serum albumin was added, and the mixture was allowed to stand at 4 ° C. for 16 hours. After washing 5 times with PBS-Tw20, 100 μl of serially diluted non-A non-B hepatitis patient serum or normal human serum
1 was added and reacted at room temperature for 1 hour. After washing 5 times with PBS-Tw20, 100,000 diluted alkaline phosphatase-conjugated anti-human IgG antibody (manufactured by TAGO) was used.
μl was added and reacted at room temperature for 1 hour. After washing each well in the same manner, 200 μl of substrate solution (1 mg / ml p-nitrophenyl phosphate) was added, and the mixture was allowed to stand in the dark for 30 minutes, and then the absorbance at 405 nm (A405) was measured. It was measured. A405, which is twice the average value of A405 in normal human serum, was used as a cutoff value, and a sample showing a value higher than that was determined to be positive. Table 1 shows the endpoints of positive sera (dilution degree of sera serving as a cutoff value) as dilution ratios.

[Table 1]

As described above, it was found that, in the normal human group, no positive person was found, whereas in the non-A non-B hepatitis patient group, the positive rate was as high as 100% and the detection was possible with high sensitivity. This allows the fusion protein of the invention to be non-A
It was shown to be specific for non-hepatitis B.

(C) Latex Agglutination Reagent Using Fusion Protein as Antigen The acrylamide latex particles described in JP-A-1-163663 are stained with 2.5% of red violet, and tannic acid is used in Example 3 ( The fusion protein C83Z3654 obtained in C) was supported on latex particles. Latex particles carrying the fusion protein thus obtained (hereinafter referred to as sensitized particles) are
Wash twice with S-0.01% sarcosinate, dilute buffer (10 mM Tris-HCl (pH 7.5), 0.15
M NaCl, 0.01% sarcosinate, 0.1% B
SA) was adjusted to 1% (w / v).

25 μl of 1% sensitized particles was added to each well of a PK plate for sheep blood cells (Olympus Optical Co., Ltd.).
In addition, 25 µl of normal human serum or blood-transmitted non-A non-B hepatitis patient serum diluted 8-fold with a dilution buffer
In addition, it was left to stand at room temperature for 1 hour. After 1 hour, the presence or absence of an agglutination image of the sensitized particles in each well was observed. As a result, an agglutination pattern was observed in the blood-transmitting non-A non-B hepatitis patient serum, but not in a normal human serum. This indicates that the fusion protein of the present invention is useful as an antigen for a latex agglutination reagent.

[0049]

[Sequence list]

 SEQ ID NO: 1 Sequence Length: 570 Sequence Type: Nucleic Acid Number of Strands: Double Strand Topology: Linear Sequence Type: Genomic DNA Origin Organism Name: Bloodborne Non-A Non-B Hepatitis Virus Sequence ATG AGC ACG AAT CCT AAA CCT CAA AGA AAA ACC AAA CGT AAC ACC AAC 48 Met Ser Thr Asn Pro Lys Pro Gln Arg Lys Thr Lys Arg Asn Thr Asn 1 5 10 15 CGC CGC CCA CAG GAC GTC AAG TTC CCG GGC GGT GGT CAG ATC GTT GGT 96 Arg Arg Pro Gln Asp Val Lys Phe Pro Gly Gly Gly Gln Ile Val Gly 20 25 30 GGA GTT TAC CTG TTG CCG CGC AGG GGC CCC AGG TTG GGT GTG CGC GCA 144 Gly Val Tyr Leu Leu Pro Arg Arg Gly Pro Arg Leu Gly Val Arg Ala 35 40 45 ACT AGG AAG ACT TCC GAG CGG TCG CAG CCT CGT GGA AGG CGA CAA CCT 192 Thr Arg Lys Thr Ser Glu Arg Ser Gln Pro Arg Gly Arg Arg Gln Pro 50 55 60 ATC CCC AAG GCT CGC CGG CCC GAG GGC AGG GCC TGG GCT CAG CCC CGG 240 Ile Pro Lys Ala Arg Arg Pro Glu Gly Arg Ala Trp Ala Gln Pro Arg 65 70 75 80 TAC CCT TGG CCC CTC TAT GGC AAT GAG GGT CTG GGG TGG GCA GGA TGG 288 Tyr Pro Trp Pro Leu Tyr Gly Asn Glu Gly Leu Gly Trp Ala Gly Trp 85 90 95 CTC CTG TCA CCC CGA GGC TCT CGG CCT AGT TTG TCA CCC ACG GAC CCC 336 Leu Leu Ser Pro Arg Gly Ser Arg Pro Ser Leu Ser Pro Thr Asp Pro 100 105 110 CGG CGT AGG TCG CGT AAT TTG GGT AAG GTC ATC GAT ACC CTT ACA TGC 384 Arg Arg Arg Ser Arg Asn Leu Gly Lys Val Ile Asp Thr Leu Thr Cys 115 120 125 GGC TTC GGA GCC CTC ATG GGG TAC ATT CCG CTC GTC GGC GCC CCC CTA 432 Gly Phe Gly Ala Leu Met Gly Tyr Ile Pro Leu Val Gly Ala Pro Leu 130 135 140 GGC ATG GGC CCC AGG GCC CTG CGG CAT GGC GTC CGG CGT CTG GAG GAC 480 Gly Met Gly Pro Arg Ala Leu Arg His Gly Val Arg Arg Leu Glu Asp 145 150 155 160 GGC GTG AAC TAC GCA ACA GGG AAT CTG CCC GGT TGC TCT TTC TCT ATC 528 Gly Val Asn Tyr Ala Thr Gly Asn Leu Pro Gly Cys Ser Phe Ser Ile 165 170 175 TTC CTT CTA GCT TTG CTG TCT TGT CTG ACC ATC CCA GCT TCC 570 Phe Leu Leu Ala Leu Leu Ser Cys Leu Thr Ile Pro Ala Ser 180 185 190

[0050]

[Sequence list]

 SEQ ID NO: 2 Sequence Length: 120 Sequence Type: Nucleic Acid Number of Strands: Double-Stranded Topology: Linear Sequence Type: Other Nucleic Acid Synthetic DNA Sequence GTG GTG CTG TCC GGC AAG CCG GCG ATC ATT CCG GAC CGC GAA GTC CTG 48 Val Val Leu Ser Gly Lys Pro Ala Ile Ile Pro Asp Arg Glu Val Leu 1 5 10 15 TAC CGC GAA TTC GAT GAG ATG GAA GAG TCC TCT CAG CAC TTA CCG TAC 96 Tyr Arg Glu Phe Asp Glu Met Glu Glu Ser Ser Gln His Leu Pro Tyr 20 25 30 ATC GAG CAA GGC ATG ATG CTG GCC 120 Ile Glu Gln Gly Met Met Leu Ala 35 40

[0051]

[Sequence list]

 SEQ ID NO: 3 Sequence Length: 846 Sequence Type: Nucleic Acid Number of Strands: Double-Stranded Topology: Linear Sequence Type: Other Nucleic Acid Hybrid DNA Sequence ATG AGC ACG AAT CCT AAA CCT CAA AGA AAA ACC AAA CGT AAC ACC AAC 48 Met Ser Thr Asn Pro Lys Pro Gln Arg Lys Thr Lys Arg Asn Thr Asn 1 5 10 15 CGC CGC CCA CAG GAC GTC AAG TTC CCG GGC GGT GGT CAG ATC GTT GGT 96 Arg Arg Pro Gln Asp Val Lys Phe Pro Gly Gly Gly Gln Ile Val Gly 20 25 30 GGA GTT TAC CTG TTG CCG CGC AGG GGC CCC AGG TTG GGT GTG CGC GCA 144 Gly Val Tyr Leu Leu Pro Arg Arg Gly Pro Arg Leu Gly Val Arg Ala 35 40 45 ACT AGG AAG ACT TCC GAG CGG TCG CAG CCT CGT GGA AGG CGA CAA CCT 192 Thr Arg Lys Thr Ser Glu Arg Ser Gln Pro Arg Gly Arg Arg Gln Pro 50 55 60 ATC CCC AAG GCT CGC CGG CCC GAG GGC AGG GCC TGG GCT CAG CCC CGG 240 Ile Pro Lys Ala Arg Arg Pro Glu Gly Arg Ala Trp Ala Gln Pro Arg 65 70 75 80 TAC CCT ATG GCC ATC ATA CGT TAT ATA GCT GAC AAG CAC AAC ATG TTG 288 Tyr Pro Met Ala Ile Ile Arg Tyr Ile Ala Asp Lys His Asn Met Leu 85 90 95 GGT GGT TGT CCA AAA GAG CGT GCA GAG ATT TCA ATG CTT GAA GGA GCG 336 Gly Gly Cys Pro Lys Glu Arg Ala Glu Ile Ser Met Leu Glu Gly Ala 100 105 110 GTT TTG GAT ATT AGA TAC GGT GTT TCG AGA ATT GCA TAT AGT AAA GAC 384 Val Leu Asp Ile Arg Tyr Gly Val Ser Arg Ile Ala Tyr Ser Lys Asp 115 120 125 TTT GAA ACT CTC AAA GTT GAT TTT CTT AGC AAG CTA CCT GAA ATG CTG 432 Phe Glu Thr Leu Lys Val Asp Phe Leu Ser Lys Leu Pro Glu Met Leu 130 135 140 AAA ATG TTC GAA GAT CGT TTA TGT CAT AAA ACA TAT TTA AAT GGT GAT 480 Lys Met Phe Glu Asp Arg Leu Cys His Lys Thr Tyr Leu Asn Gly Asp 145 150 155 160 CAT GTA ACC CAT CCT GAC TTC ATG TTG TAT GAC GCT CTT GAT GTT GTT 528 His Val Thr His Pro Asp Phe Met Leu Tyr Asp Ala Leu Asp Val Val 165 170 175 TTA TAC ATG GAC CCA ATG TGC CTG GAT GCG TTC CCA AAA TTA GTT TGT 576 Leu Tyr Met Asp Pro Met Cys Leu Asp Ala Phe Pro Lys Leu Val Cys 180 185 190 TTT AAA AAA CGT ATT GAA GCT ATC CCA CAA ATT GAT AAG TAC TTG AAA 624 Phe Lys Lys Arg Ile Glu Ala Ile Pro Gln Ile Asp Lys Tyr Leu Lys 195200 205 TCC AGC AAG TAT ATA GCA TGG CCT TTG CAG GGC TGG CAA GCC ACG TTT 672 Ser Ser Lys Tyr Ile Ala Trp Pro Leu Gln Gly Trp Gln Ala Thr Phe 210 215 220 GGT GGT GGC GAC CAT CCT CCA AAA TCG GAT CTG GTT CCG CGT GGA TCC 720 Gly Gly Asp His Pro Pro Lys Ser Asp Leu Val Pro Arg Gly Ser 225 230 235 240 GTG GTG CTG TCC GGC AAG CCG GCG ATC ATT CCG GAC CGC GAA GTC CTG 768 Val Val Leu Ser Gly Lys Pro Ala Ile Ile Pro Asp Arg Glu Val Leu 245 250 255 TAC CGC GAA TTC GAT GAG ATG GAA GAG TCC TCT CAG CAC TTA CCG TAC 816 Tyr Arg Glu Phe Asp Glu Met Glu Glu Ser Ser Gln His Leu Pro Tyr 260 265 270 ATC GAG CAA GGC ATG ATG CTG GCC AAT TAA 846 Ile Glu Gln Gly Met Met Leu Ala Asn *** 275 280

[0052]

[Sequence list]

 SEQ ID NO: 4 sequence length: 1260 sequence type: nucleic acid number of strands: double-stranded topology: linear sequence type: Genomic DNA origin organism name: blood-borne non-A non-B hepatitis virus sequence CAT- 1 ATG AGC ACG AAT CCT AAA CCT CAA AGA AAA ACC AAA CGT AAC ACC AAC 48 Met Ser Thr Asn Pro Lys Pro Gln Arg Lys Thr Lys Arg Asn Thr Asn 1 5 10 15 CGC CGC CCA CAG GAC GTC AAG TTC CCG GGC GGT GGT CAG ATC GTT GGT 96 Arg Arg Pro Gln Asp Val Lys Phe Pro Gly Gly Gly Gln Ile Val Gly 20 25 30 GGA GTT TAC CTG TTG CCG CGC AGG GGC CCC AGG TTG GGT GTG CGC GCA 144 Gly Val Tyr Leu Leu Pro Arg Arg Gly Pro Arg Leu Gly Val Arg Ala 35 40 45 ACT AGG AAG ACT TCC GAG CGG TCG CAG CCT CGT GGA AGG CGA CAA CCT 192 Thr Arg Lys Thr Ser Glu Arg Ser Gln Pro Arg Gly Arg Arg Gln Pro 50 55 60 ATC CCC AAG GCT CGC CGG CCC GAG GGC AGG GCC TGG GCT CAG CCC CGG 240 Ile Pro Lys Ala Arg Arg Pro Glu Gly Arg Ala Trp Ala Gln Pro Arg 65 70 75 80 TAC CCT TGG CCC CTC TAT GGC AAT GAG GGT CTG GGG TGG GCA GGA TGG 288 Tyr Pro Trp P ro Leu Tyr Gly Asn Glu Gly Leu Gly Trp Ala Gly Trp 85 90 95 CTC CTG TCA CCC CGA GGC TCT CGG CCT AGT TTG TCA CCC ACG GAC CCC 336 Leu Leu Ser Pro Arg Gly Ser Arg Pro Ser Leu Ser Pro Thr Asp Pro 100 105 110 CGG CGT AGG TCG CGT AAT TTG GGT AAG GTC ATC GAT ACC CTT ACA TGC 384 Arg Arg Arg Ser Arg Asn Leu Gly Lys Val Ile Asp Thr Leu Thr Cys 115 120 125 GGC TTC GGA GCC CTC ATG GGG TAC ATT CCG CTC GTC GGC GCC CCC CTA 432 Gly Phe Gly Ala Leu Met Gly Tyr Ile Pro Leu Val Gly Ala Pro Leu 130 135 140 GGC ATG GGC CCC AGG GCC CTG CGG CAT GGC GTC CGG CGT CTG GAG GAC 480 Gly Met Gly Pro Arg Ala Leu Arg His Gly Val Arg Arg Leu Glu Asp 145 150 155 160 GGC GTG AAC TAC GCA ACA GGG AAT CTG CCC GGT TGC TCT TTC TCT ATC 528 Gly Val Asn Tyr Ala Thr Gly Asn Leu Pro Gly Cys Ser Phe Ser Ile 165 170 175 TTC CTT CTA GCT TTG CTG TCT TGT CTG ACC ATC CCA GCT TCC GCT TAC 576 Phe Leu Leu Ala Leu Leu Ser Cys Leu Thr Ile Pro Ala Ser Ala Tyr 180 185 190 GAA GTG CGC AAC GAG TCC GGG GTG TAC CAT GTC ACG AAC GAC TGC TCC 624 Glu Val Arg Asn Glu Ser Gly Val Tyr His Val Thr Asn Asp Cys Ser 195 200 205 AAC TCA AGT ATT GTG TAT GAG GCA GCG GAC ATG ATC ATG CAA CCC CCC 672 Asn Ser Ser Ile Val Tyr Glu Ala Ala Asp Met Ile Met Gln Pro Pro 210 215 220 GGG TGC GTG CCC TGC ATC CGA GAG AAC AAT TCC TCC CGT TGC TGG GTG 720 Gly Cys Val Pro Cys Ile Arg Glu Asn Asn Ser Ser Arg Cys Trp Val 225 230 235 240 GCG CTC ACC CCC ACG CTC GCG GCC AGG AAC AAC AGC ATC CCC ACC ACG 768 Ala Leu Thr Pro Thr Leu Ala Ala Arg Asn Asn Ser Ile Pro Thr Thr 245 250 255 ACA ATA CGA CGC CAC GGA TCT TTG CTT GTT GGG GCA GCT GCT CTC TGT 816 Thr Ile Arg Arg His Gly Ser Leu Leu Val Gly Ala Ala Ala Leu Cys 260 265 270 TCC GCT ATG TAC GTA GGG GAT CTC TGC GGA TCT GTC TTC CTC GTC TCC 864 Ser Ala Met Tyr Val Gly Asp Leu Cys Gly Ser Val Phe Leu Val Ser 275 280 285 CAG CTG TTC ACC TTC TCA CCT CGC CGG TAT GAG ACG GTA CAA GAA TGC 912 Gln Leu Phe Thr Phe Ser Pro Arg Arg Tyr Glu Thr Val Gln Glu Cys 290 295 300 AAC TGC TCA CTC TAT CCC GGC CAC GTA TCA GGT CAC CGC ATG GCTTGG 960 Asn Cys Ser Leu Tyr Pro Gly His Val Ser Gly His Arg Met Ala Trp 305 310 315 320 GAT ATG ATG ATG AAC TGG TCA CCT ACA ACG GCC CTA GTG GTA TCG CAG 1008 Asp Met Met Met Asn Trp Ser Pro Thr Thr Ala Leu Val Val Ser Gln 325 330 335 TTA CCC GGA TCC CAC AAG CCG TCG TGG ATA TGG TGG CGG GGC CAC TGG 1056 Leu Pro Gly Ser His Lys Pro Ser Trp Ile Trp Trp Arg Gly His Trp 340 345 350 GGA GTC CTA GCG GGC CTT GCC TAC TAT TCC ATG GTG GGG AAC TGG GCT 1104 Gly Val Leu Ala Gly Leu Ala Tyr Tyr Ser Met Val Gly Asn Trp Ala 355 360 365 AAA GTG TTG GTT GTG ATG CTA CTC TTT GCC GGT GTT AGC GGC TCG TCT 1152 Lys Val Leu Val Val Met Leu Leu Phe Ala Gly Val Ser Gly Ser Ser 370 375 380 ACC TAC ACG ACA GGG GGA CAA GTG GGT CGC CAG ACT AGT CGT CAC GGG 1200 Thr Tyr Thr Thr Gly Gly Gln Val Gly Arg Gln Thr Ser Arg His Gly 385 390 395 400 GCC CTC TTC AGC CAG GGG GCG TCT CAG GAA GTC CAA CTT GTG AAT ACC 1248 Ala Leu Phe Ser Gln Gly Ala Ser Gln Glu Val Gln Leu Val Asn Thr 405 410 415 AAC GGC AGC GGG 1260 Asn Gly Ser 419

─────────────────────────────────────────────────── ─── Continuation of front page (51) Int.Cl. ⁵ Identification code Internal reference number FI Technical display location G01N 33/53 D 8310-2J 33/576 Z 9015-2J // C12N 15/51 ZNA 15/62 C12P 21/02 C 8214-4B (C12P 21/02 C12R 1:19) (72) Inventor Masatoshi Nagauchi 2-43-2 Hatagaya, Shibuya-ku, Tokyo Olympus Optical Co., Ltd. (72) Inventor Jericho Mori 2-43-2 Hatagaya, Shibuya-ku, Tokyo Olympus Optical Co., Ltd. (72) Inventor Kyoko Kaneko 2-43-2 Hatagaya, Shibuya-ku, Tokyo Olympus Optical Co., Ltd. (72) Makoto Nakamura Tokyo 2-43-2 Hatagaya, Shibuya-ku, Tokyo Olympus Optical Industry Co., Ltd.

Claims (3)

[Claims] 1. A peptide (A) containing at least an epitope of a core protein derived from blood-borne non-A non-B hepatitis virus and a peptide (B) containing at least an epitope of another protein have isoelectric points in the acidic region. It is connected through the carrier protein (C) it has, and has an isoelectric point of 6 to
10. A fusion protein having a molecular weight of 25 to 100 kilodaltons. 2. The peptide (B) is a nonstructural protein NS
4. The fusion protein according to claim 1, which is a part of 4. 3. The fusion protein according to claim 2, wherein the epitope of the core protein and the epitope of the nonstructural protein NS4 are the amino acid sequences of SEQ ID NOs: 1 and 2, respectively.