JPH05310786A - Peptide of hcv envelope region - Google Patents

Abstract

PURPOSE:To obtain the peptide useful for detecting hepatitis C vaccine(HCV) or for vaccine, reacting to an antibody against HCV, containing a specific amino acid, capable of detecting HCV in a precritical state regardless of type of HCV. CONSTITUTION:A protected peptide is synthesized from t-butoxycarbonyl(t- BOC)amino acid symmetrical anhydride by using an automatic peptide synthesizing device and then the protected peptide is dissolved in a mixed solution of anisole, dimethyl sulfite and p-thiocresol, reacted and deprotected in the presence of hydrogen fluoride at 0-5 deg.C for 1 hour. The prepared crude crystal is dissolved in 2N acetic acid, extracted, freeze-dried and purified by liquid reversed phase chromatography to give the objective peptide having reactivity to an antibody against hepatitis C virus, comprising at least six amino acids, containing a sequence coincident with part of amino acid sequence of formula I or formula II.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a peptide corresponding to a part of the antigenic site of a protein encoded by the envelope region of hepatitis C virus gene. The peptide of the present invention can be used for detecting an antibody against hepatitis C virus, and is administered to mammals for the purpose of obtaining a polyclonal antibody or a monoclonal antibody against a protein encoded by the envelope region of the hepatitis C virus gene. It can be used as an immunogen, a vaccine administered for the purpose of preventing and treating hepatitis C virus infection, and the like.

[0002]

2. Description of the Related Art Causes of hepatitis include those caused by viruses, alcohol and drugs. Of these, hepatitis caused by viruses has a large proportion.

Several viruses are currently known as hepatitis-causing viruses, such as orally infected hepatitis A virus (hereinafter abbreviated as HAV) and hepatitis B virus (hereinafter abbreviated as HBV) that is infected by blood transfusion. There is.

HBV is known as one of the causes of hepatitis (post-transfusion hepatitis) that occurs after blood transfusion, but many post-transfusion hepatitis not caused by HBV has also occurred. In Japan,
Although the number of HBV-infected persons due to blood transfusion is drastically reduced by eliminating the blood containing HBV from the blood for transfusion, posttransfusion hepatitis patients are not endless. The cause of this has remained unknown for a long time and was conventionally called non-A non-B hepatitis.

In recent years, Choo, QL et al. (Science 24
4, 359-362, 1989) and Kuo, G. et al. (Science 244, 362-3).
62, 1989) revealed the presence of non-A non-B hepatitis virus. This causes post-transfusion hepatitis,
They call it hepatitis C virus (hereinafter abbreviated as HCV).

A method for separating HCV is disclosed in Japanese Patent Laid-Open No. 2-50088.
No. 0 (International Publication No. WO89 / 04669). HCV has been known to infect humans and chimpanzees only before the virus was identified. Therefore, in Japanese Unexamined Patent Publication No. 2-500880, HCV is separated by the following procedure.

First, the same preparation as the blood coagulation factor VIII preparation which is considered to have caused non-A non-B hepatitis is administered to a chimpanzee to develop non-A non-B hepatitis in the chimpanzee. Next, this chimpanzee liver extract is administered to another chimpanzee to develop non-A non-B hepatitis in the same manner. The blood pressure obtained from this chimpanzee is administered to another chimpanzee to confirm the onset of non-A non-B hepatitis. Concentration of virus particles assuming that the target virus is flavivirus in the blood and blood that was confirmed to develop (Bradeley, DW et al., Gastroenterology 88, 773-7
79, 1989) to extract RNA. CDNA is synthesized from this RNA to prepare a λgt11 library. Screening this using non-A non-B hepatitis convalescent chimpanzee serum and non-A non-B chronic hepatitis patient serum,
Select the clones to react.

[0008] In Japanese Patent Laid-Open No. 2-500880, as a result of these operations, it was revealed that the clone named 5-1-1 was a cDNA derived from HCV, and the gene sequence of HCV was also revealed. ..

On the other hand, the present applicant has filed Japanese Patent Application No. 2-27961.
In No. 3, a peptide which is a part of the non-structural region to which the above-mentioned clone belongs is named mokk-2 + mokk-3, and a diagnostic reagent for HCV antibody with high detection rate is prepared.

Furthermore, the applicant of the present invention has filed Japanese Patent Application No. 2-2824.
In No. 31, a partial peptide of the core protein, which is a structural region in which the amino acid sequence is highly conserved, was selected and named mokk-c1.
A diagnostic reagent for CV antibody is being produced.

[0011]

However, in the method disclosed in Japanese Patent Application Laid-Open No. 2-500880, anti-HC
For the detection of V antibody, a fusion protein in which a part of the human superoxide dismutase gene containing 5-1-1 is expressed in yeast, that is, recombinant protein C100-3 is used. This C100-3 is a nonstructural protein of HCV, and is not a protein that constitutes structural proteins such as core and envelope. Therefore, antibodies against this protein do not function as so-called neutralizing antibodies that eliminate the virus from the body. Antibodies against the protein encoded by the C100-3 region were used 6 months after HCV infection.
It will not appear until more than a month has passed. In addition, the antibody may not be detected even after the onset. In addition to this, since it contains a part of human superoxide dismutase,
A non-specific reaction to this may occur.

[0012] Recently, it has been known that HCV is roughly divided into two subspecies, that is, Western type and Asian type (Kato, N., Ohkoshi, S., Shimotohno, K .:
Proc. Jpn. Acad., 65B, 219-223, 1989). As a result of analyzing the HCV gene (RNA), the nucleotide sequences between the Western type and the Asian type are different, and the gap is large.
It was revealed to be another subspecies. In particular, it has been found that mutations in the region that is thought to encode the envelope protein are significant. In other words, the HCV envelope proteins differ greatly in amino acid sequence depending on the species, and the epitopes having antigenicity also differ. To immunologically confirm the presence of antibodies to the envelope protein, prepare a polyprotein artificially synthesized by gene recombination technology or peptide synthesis technology according to the gene encoding the envelope according to each virus species. There must be. Also,
Similarly, when using the above polyprotein as a vaccine, it must be prepared according to each virus species.

H. Okamoto et al., Japanese Journ
In al of Experimental Medicine 60, 167-177, 1990, considering the various mutations of HCV,
We have calculated the peptides of highly conserved amino acid regions in the region, and report that these regions will be useful for HCV classification, infection diagnosis and vaccine development.

As described above, when the anti-HCV antibody is analyzed using the conventional C100-3 antigen, only about 70% of the patients infected with HCV can be detected, and a non-specific reaction may occur. In addition, C100-3 region and above mokk-2 + m
Since the protein encoded by okk-3 is a nonstructural protein of the virus, antibodies against it have no effect on eliminating the virus from the body. Therefore, the protein encoded by the C100-3 region and the above mokk-2 + mokk-3 cannot be expected to be effective as a vaccine.

Further, when a region having a highly conserved amino acid sequence is used, a common reactivity can be expected with respect to the above-mentioned various variability, but a high detection rate cannot be expected. There is.

The present invention provides a peptide corresponding to the antigenic site of the envelope region of HCV, which can be used for detecting anti-HCV antibodies present in the serum of HCV-infected patients and can also be used as a vaccine. With the goal. Another object of the present invention is to provide a reagent and an effective vaccine capable of maintaining a high detection rate for various HCV mutations.

[0017]

[Means and Actions for Solving the Problems] In view of the present situation, as a result of intensive investigations by the inventors, as a result, amino acid regions having high reactivity in common in each of the different subspecies groups are actually different groups. -The hypothesis that it exists in a region outside the highly conserved region within the group. That is, in order to complete the present invention, the amino acid region that is highly mutated in each group is the one that has the uniquely high reactivity among the various variability. I arrived. Geoffrey Rs (Nature 302, 490-49
5, 1983) have revealed that the synthesized peptide has antigenicity to the antibody.

The inventors of the present invention have made it clear that the H
Based on the amino acid sequence obtained from the gene sequence of the envelope region of CV, a site having strong antigenicity is predicted from the hydrophobicity value of each amino acid, and a peptide having the amino acid sequence is synthesized to confirm the antigenicity. I have reached the goal.

That is, the peptide of the present invention is a peptide having reactivity with an antibody against hepatitis C virus, which peptide has the amino acid sequence shown in SEQ ID NO: 1 or SEQ ID NO: 2 in the sequence listing below. It is characterized in that it comprises a sequence that matches a part and that consists of at least 6 amino acids.

The peptide of the present invention preferably has a sequence in its sequence which corresponds to all of the amino acid sequences shown in SEQ ID NO: 1 or 2 in the sequence listing shown below. Also, a peptide corresponding to a sequence consisting of at least 6 amino acids in the amino acid sequence shown in SEQ ID NO: 1 or 2 is within the scope of the present invention. The fact that the minimum size of an antigenic determinant is a sequence consisting of 6 amino acids means that Immunochemistry 12, 423-438, 197
5, and Proc. Natl. Acad. USA, vol.78, 3824-382.
8 clarified.

Therefore, even a part of the peptide having the sequence shown in SEQ ID NO: 1 or 2 is composed of a sequence consisting of 6 or more consecutive amino acids and has a cross-reactivity with the peptide having the above sequence. Are included in this invention.

The analogue of the peptide specified by the amino acid sequence shown in SEQ ID NO: 1 or 2 means that one or more amino acids in the amino acid sequence shown in SEQ ID NO: 1 or 2 are replaced with another amino acid. , Deleted, and a peptide having one or more amino acids added to the amino acid sequence shown in SEQ ID NO: 1 or 2 and having cross-reactivity with the peptide shown in SEQ ID NO: 1 or 2. .. Whether or not it has cross-reactivity can be confirmed by testing the immunoreactivity to the antibody against HCV due to the structure of the amino acid sequence.

The peptide of the present invention can be immobilized on various carriers and analyzed for anti-HCV antibody. Examples of the carrier used here include bovine serum albumin, polypeptides, test tubes and microplates made of polystyrene, glass, etc., or balls.

[0024]

【Example】

 Example 1: Production of peptides

Genes in the envelope region are largely different depending on virus species or change due to mutation. For this reason, as described above, it is said that HCV is roughly divided into species of Asian type and Western type.

Western type HCV is shown in FIG. 1 and Asian type HC is shown in FIG.
The amino acid sequence of the envelope region representing each of V is shown. The present inventors calculated the antigenic site based on this. That is, each amino acid is given a unique hydrophilicity value,
The hydrophilicity values of 6 consecutive amino acids in the amino acid sequences of FIGS. 1 and 2 are summed up to obtain the hydrophilicity value of the peptide consisting of the 6 amino acids. This operation is shown in the HC
All combinations in the amino acid sequence of the V primary structure were performed. The results are shown in FIG. 3 only for Western type HCV. In the figure, the horizontal axis represents the position of the amino acid, from N-terminal to C-terminal from left to right. The vertical axis represents the hydrophilicity value of a peptide consisting of 6 consecutive amino acids. The higher the hydrophilicity, the higher the susceptibility to recognition as an antigen by the immune system.

From FIG. 3, a peptide located in the envelope region of HCV called Western type and predicted to have high antigenicity was selected and designated as EP1. This is the amino acid sequence of the HCV envelope region shown in FIG.
It has the amino acid sequence corresponding to the 30th to 260th positions and shown as SEQ ID NO: 1 in the sequence listing below. Similarly,
A peptide predicted to have high antigenicity was selected from the Asian HCV envelope region and designated as EP2. This corresponds to the 230-260th position in the amino acid sequence representing the envelope region of Asian HCV shown in FIG. 2 and has the amino acid sequence shown as SEQ ID NO: 2 in the sequence listing below.

Next, the peptides EP1 and EP2 in the envelope region of HCV were synthesized by the following method. First, Applied Biosystems 430A Peptide Synthsiz
The peptide was synthesized with a symmetrical anhydride of t-butoxycarbonyl (t-BOC) amino acid. Then, this peptide was dissolved in a mixed solution of anisole, dimethylsulfite, and parathiocresol, and reacted at 0-5 ° C for 1 hour in the presence of hydrogen fluoride for deprotection.
This method is based on S. Sakakibara (Bull. Chem. Soc. Jpn. 40,
2164, 1967). 2N of the obtained crude crystals
It was dissolved in acetic acid, extracted, and freeze-dried. This was purified by liquid reverse phase chromatography and then freeze-dried again. Example 2: Anti-H from a blood donor sample by enzyme immunoassay
Detection of CV antibody

Peptide EP1 prepared in Example 1
Using EP2 and EP2, anti-HCV antibody contained in the serum (blood) of the HCV infected person was detected by enzyme immunoassay (EIA). (A) Preparation of peptide-immobilized microwell plate

Peptide EP1 prepared in Example 1
EP2 and EP2 were dissolved in phosphate buffered saline (PBS), dispensed into separate wells of a 96-well microwell plate manufactured by NUNC, and reacted at room temperature for 1 hour. Then, the solution in the well was removed, and the inner wall of the well was washed 3 times with PBS. Next, PBS containing 1% gelatin was dispensed and reacted at room temperature for 1 hour. Then, the solution in the well was removed, and the inner wall of the well was washed 3 times with PBS. This was dried at room temperature to obtain a peptide solid-phased microwell plate. (B) Sample measurement

The donated 500 specimens were diluted with PBS containing bovine serum albumin (BSA) and Tween 20, and then dispensed to the peptide-immobilized microplate prepared in (a), and the mixture was incubated at 37 ° C. for 30 minutes. Let react for minutes. Then Tween
The inner wall of the well was washed 3 times with PBS containing 20 times. Next, a 1000-fold diluted peroxidase-labeled goat anti-human IgG was dispensed and allowed to react at 37 ° C for 30 minutes, followed by Tweezers.
The inner wall of the well was washed 6 times with PBS containing n 20. Further, a citrate buffer solution containing hydrogen peroxide and o-phenylenediamine was dispensed and reacted at 37 ° C. for 20 minutes, and then 5N sulfuric acid was dispensed to stop the reaction. The absorbance of this was measured using a plate reader. The results are shown in Table 1 below.

[0032]

[Table 1]

From this, among the 500 donated blood samples, E
Nine specimens react with P1, and specimens react with EP2
It was found that there are 19 specimens, and among these, 3 specimens that react with both EP1 and EP2 exist.

The measurement was carried out in the same manner as in (a) and (b) above using the peptides mokk-2 + mokk-3 and mokk-c1 which had been already disclosed by the applicant, and the results were the same as above. Of the 500 samples, only 3 tested positive. From this, it is considered that the reagent based on the peptide of the present invention shows a high detection rate for two types of HCV subspecies roughly classified. Example 3: Detection of anti-HCV antibody from non-A non-B hepatitis patient specimens by EIA

Using the peptide-immobilized Mike Twell plate prepared in the same manner as in Example 2 (a), Example 2
The same measurement operation as in (b) was performed to detect an antibody against the HCV envelope region peptide in a hepatitis patient sample. The results are shown in Table 2 below.

[0036]

[Table 2]

As is clear from the table, among 32 cases of acute hepatitis, 3 cases responded to EP1 and 3 cases responded to EP2. From 28 cases of chronic hepatitis patients
No case was found to react with any of the two peptides. EP from 15 patients with cirrhosis
One case responding to 1 was found, and no case responding to EP2 was found. And patients with hepatocellular carcinoma
Out of 16 cases, 4 cases responding to EP1 and 4 cases responding to EP2 were found. Example 4: Time course of anti-HCV antibody in a sample of acute non-A non-B hepatitis patient after blood transfusion by EIA

Using the peptide-immobilized microwell plate prepared in the same manner as in Example 2 (a), Example 2 (b) was used.
Perform the same measurement procedure as above to determine the H
The time course of the antibody against the CV envelope region peptide was examined. The result is shown in FIG.

As is clear from FIG. 4, GPT increased and the symptom worsened at the same time as EP1 and EP.
Antibodies to 2 were produced and then remitted. this is,
HC produced by the production of antibodies to the envelope
This indicates that V was neutralized and remitted, and that the anti-envelope antibody is a neutralizing antibody.

[0040]

As described above, the peptide of the present invention is
It is based on the amino acid sequences that represent HCV species when they are divided into two types, and has high reactivity regardless of the diversity of each type of virus. Therefore, it is possible to prevent omission of diagnosis when detecting HCV infection.

Since the peptide of the present invention is selected from the envelope region of HCV, the anti-envelope antibody to be detected is a neutralizing antibody for this peptide. Therefore, it is possible to detect even when HCV is infected but does not develop.

Furthermore, since the anti-envelope antibody can neutralize HCV, the antibody produced by inoculating the peptide of the present invention as an antigen can neutralize HCV. Therefore, the peptide of this invention can be used as a vaccine. Since the peptide of this invention is a part of the envelope region and can be obtained by synthesis, it is possible to produce a vaccine very safely.

[0043]

[Sequence list]

 Sequence number: 1 Array length: 31 Array type: Amino Acid Sequence Val Arg Glu Gly Asn Val Ser Arg Cys Trp Val Ala Met Thr Pro 1 5 10 15 Thr Val Ala Thr Arg Asp Gly Lys Leu Pro Ala Thr Gln Leu Arg 20 25 30 Arg SEQ ID NO: 2 Array length: 31 Array type: Amino Acid Sequence Val Arg Glu Asp Asn Ser Ser Arg Cys Trp Val Ala Leu Thr Pro 1 5 10 15 Thr Leu Ala Ala Arg Asn Ala Ser Val Pro Thr Thr Thr Ile Arg 20 25 30 Arg SEQ ID NO: 3 Array length: 319 Array type: Amino acid sequence Tyr Gln Val Arg Asn Ser Thr Gly Leu Tyr His Val Thr Asn Asp 1 5 10 15 Cys Pro Asn Ser Ser Ile Val Tyr Glu Ala His Asp Ala Ile Leu 20 25 30 His Thr Pro Gly Cys Val Pro Cys Val Arg Glu Gly Asn Val Ser 35 40 45 Arg Cys Trp Val Ala Met Thr Pro Thr Val Ala Thr Arg Asp Gly 50 55 60 Lys Leu Pro Ala Thr Gln Leu Arg Arg His Ile Asp Leu Leu Val 65 70 75 Gly Ser Ala Thr Leu Cys Ser Ala Leu Tyr Val Gly Asp Leu Cys 80 85 90 Gly Ser Val Phe Leu Ile Gly Gln Leu Phe Thr Phe Ser Pro Arg 95 100 105 Arg His Trp Thr Thr Gln GIy Cys Asn Cys Ser Ile Tyr Pro Gly 110 115 120 His Ile Thr Gly His Arg Met Ala Trp Asp Met Met Met Asn Trp 125 130 135 Ser Pro Thr Ala Ala Leu Val Met Ala Gln Leu Leu Arg Ile Pro 140 145 150 Gln Ala Ile Leu Asp Met Ile Ala Gly Ala His Trp Gly Val Leu 155 160 165 Ala Gly Ile Ala Tyr Phe Ser Met Val Gly Asn Trp Ala Lys Val 170 175 180 Leu Val Val Leu Leu Leu Phe Ala Gly Val Asp Ala Glu Thr Ile 185 190 195 Val Ser Gly Gly Gln Ala Ala Arg Ala Met Ser Gly Leu Val Ser 20 0 205 210 Leu Phe Thr Pro Gly Ala Lys Gln Asn Ile Gln Leu Ile Asn Thr 215 220 225 Asn Gly Ser Trp His Ile Asn Ser Thr Ala Leu Asn Cys Asn Glu 230 235 240 Ser Leu Asn Thr Gly Trp Leu Ala Gly Leu Ile Tyr Gln His Lys 245 250 255 Phe Asn Ser Ser Gly Cys Pro Glu Arg Leu Ala Ser Cys Arg Arg 260 265 270 Leu Thr Asp Phe Asp Gln Gly Trp Gly Pro Ile Ser His Ala Asn 275 280 285 Gly Ser Gly Pro Asp Gln Arg Pro Tyr Cys Trp His Tyr Pro Pro 290 295 300 Lys Pro Cys Gly Ile Val Pro Ala Lys Ser Val Cys Gly Pro Val 305 310 315 Tyr Cys Phe Thr SEQ ID NO: 4 Array length: 319 Array type: Amino acid sequence Tyr Glu Val Arg Asn Val Ser Gly Ile Tyr His Val Thr Asn Asp 1 5 10 15 Cys Ser Asn Ser Ser Ile Val Tyr Glu Ala Ala Asp Met Ile Met 20 25 30 His Thr Pro Gly Cys Val Pro Cys Val Arg Glu Asp Asn Ser Ser 35 40 45 Arg Cys Trp Val Ala Leu Thr Pro Thr Leu Ala Ala Arg Asn Ala 50 55 60 Ser Val Pro Thr Thr Ile Arg Arg His Val Asp Leu Leu Val 65 70 75 Gly Ala Ala Ala Phe Cys Ser Ala Met Tyr Val Gly Asp Leu Cys 80 85 90 Gly Ser Val Phe Leu Val Ser Gln Leu Phe Thr Phe Ser Pro Arg 95 100 105 Arg His Glu Thr Val Gln Asp Cys Asn Cys Ser Ile Tyr Pro Gly 110 115 120 His Leu Ser Gly His Arg Met Ala Trp Asp Met Met Met Asn Trp 125 130 135 Ser Pro Thr Thr Ala Leu Val Val Ser Gln Leu Leu Arg Ile Pro 140 145 150 Gln Ala Val Val Asp Met Val Ala Gly Ala His Trp Gly Val Leu 155 160 165 Ala Gly Leu Ala Tyr Tyr Ser Met Val Gly Asn Trp Ala Lys Val 170 175 180 Leu Ile Val Ala Leu Leu Phe Ala Gly Val Asp Gly Glu Thr Tyr 185 190 195 Thr Ser Gly Gly Ala Ala Ser His Thr Thr Ser Thr Leu Ala Ser 20 0 205 210 Leu Phe Ser Pro Gly Ala Ser Gln Arg Ile Gln Leu Val Asn Thr 215 220 225 Asn Gly Ser Trp His Ile Asn Arg Thr Ala Leu Asn Cys Asn Asp 230 235 240 Ser Leu His Thr Gly Phe Leu Ala Ala Leu Phe Tyr Thr His Arg 245 250 255 Phe Asn Ser Ser Gly Cys Pro Glu Arg Met Ala Ser Cys Arg Pro 260 265 270 Ile Asp Trp Phe Ala Gln Gly Trp Gly Pro Ile Thr Tyr Thr Glu 275 280 285 Pro Asp Ser Pro Asp Gln Arg Pro Tyr Cys Trp His Tyr Ala Pro 290 295 300 Arg Pro Cys Gly Ile Val Pro Ala Ser Gln Val Cys Gly Pro Val 305 310 315 Tyr Cys Phe Thr

[Brief description of drawings]

FIG. 1 is a view showing an amino acid sequence of a typical Western-type HCV envelope region.

FIG. 2 is a diagram showing an amino acid sequence of a typical Asian HCV envelope region.

FIG. 3 is a graph showing the sum of hydrophilicity values of 6 consecutive amino acids in the amino acid sequence of the envelope region of the Western-type HCV shown in FIG.

FIG. 4 is a graph showing the time course of the antibody against the HCV envelope region peptide in the acute hepatitis patient sample, which was measured using a microwell plate on which the peptide of the present invention was immobilized in Example 4. ..

─────────────────────────────────────────────────── ───

[Procedure amendment]

[Submission date] January 25, 1993

[Procedure Amendment 1]

[Document name to be amended] Statement

[Correction target item name] 0009

[Correction method] Change

[Correction content]

On the other hand, the present applicant has filed Japanese Patent Application No. 2-28243.
In No. 1 , a peptide which is a part of the non-structural region to which the above-mentioned clone belongs is named mokk-33, and a diagnostic reagent for HCV antibody with high detection rate is prepared.

[Procedure Amendment 2]

[Document name to be amended] Statement

[Correction target item name] 0014

[Correction method] Change

[Correction content]

As described above, when the anti-HCV antibody is analyzed using the conventional C100-3 antigen, only about 70% of the patients infected with HCV can be detected, and a non-specific reaction may occur. Moreover, since the C100-3 region and the protein encoded by the above mokk-33 are nonstructural proteins of the virus, antibodies against them have no effect of eliminating the virus from the body. Therefore, C100-3 region and above
The protein encoded by mokk-33 cannot be expected to be effective as a vaccine.

[Procedure 3]

[Document name to be amended] Statement

[Correction target item name] 0034

[Correction method] Change

[Correction content]

When the peptides mokk-33 and mokk-c1 already disclosed by the applicant were measured in the same manner as in the above (a) and (b), the same 500 samples as above were analyzed. Only 3 specimens were scored positive. From this, it is considered that the reagent based on the peptide of the present invention shows a high detection rate for two types of HCV subspecies roughly classified. Example 3: Detection of anti-HCV antibody from non-A non-B hepatitis patient specimens by EIA

Continuation of the front page (51) Int.Cl. ⁵ Identification code Reference number within the agency FI Technical display location G01N 33/53 D 8310-2J 33/569 L 9015-2J 33/576 Z 9015-2J // C07K 99:00

Claims (5)

[Claims] 1. A peptide having reactivity with an antibody against hepatitis C virus, wherein the peptide consists of at least 6 amino acids, and has the following (a) or (b):
A peptide containing a part of the amino acid sequence represented by. (A) EP1 Val Arg Glu Gly Asn Val Ser Arg Cys Trp 10 Val Ala Met Thr Pro Thr Val Ala Thr Arg 20 Asp Gly Lys Leu Pro Ala Thr Gln Leu Arg 30 Arg 31 (b) EP2 Val Arg Glu Asp Asn Ser Ser Arg Cys Trp 10 Val Ala Leu Thr Pro Thr Leu Ala Ala Arg 20 Asn Ala Ser Val Pro Thr Thr Ile Arg 30 Arg 31 2. One or more amino acid sequences represented by the above (a) or (b) within a range in which the immunoreactivity to the antibody of hepatitis C virus due to the tertiary structure of the protein is maintained. The peptide according to claim 1, wherein an amino acid is substituted or deleted, or one or more amino acids are added. 3. The peptide according to claim 1, which is chemically synthesized within a range in which immunoreactivity to an antibody of hepatitis C virus caused by the tertiary structure of the protein is maintained. 4. A mixture of a peptide having the amino acid sequence represented by the following (a) and a peptide having the amino acid sequence represented by the following (b). (A) EP1 Val Arg Glu Gly Asn Val Ser Arg Cys Trp 10 Val Ala Met Thr Pro Thr Val Ala Thr Arg 20 Asp Gly Lys Leu Pro Ala Thr Gln Leu Arg 30 Arg 31 (b) EP2 Val Arg Glu Asp Asn Ser Ser Arg Cys Trp 10 Val Ala Leu Thr Pro Thr Leu Ala Ala Arg 20 Asn Ala Ser Val Pro Thr Thr Ile Arg 30 Arg 31 5. A hepatitis C vaccine containing the peptide according to claim 1.