JPH11124398A - Antigenic peptide derived from hepatitis c virus and antibody testing agent using the same - Google Patents

Abstract

PROBLEM TO BE SOLVED: To obtain the subject new peptide which is an antigenic site present in a protease derived from a hepatitis C virus, comprising a chain peptide containing a specific amino acid sequence and useful as an antibody testing agent or the like for HCV. SOLUTION: This new antigenic peptide is derived from a hepatitis C virus comprising >=12 amino acid residues containing at least one amino acid sequence selected from the group consisting of formulae I, II and III (Xaa is an optional amino acid) and is useful as a hepatitis C virus antibody testing agent or the like. The antigenic peptide is obtained by inserting a random peptide library composed of a polypeptide comprising 12 amino acid residues into an active site of a Thioredoxin protein derived from Escherichia coli, joining the resultant modified protein to flagella of the Escherichia coli, screening the prepared random peptide library by reactivity with an anti-HCV antibody and selecting the peptide reactive with the antibody.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

TECHNICAL FIELD The present invention relates to a peptide chain serving as an antigen site present in a protease derived from hepatitis C virus, and to a hepatitis C virus antibody test agent using the antigen peptide. Specifically, the present invention relates to a peptide chain containing a partial amino acid sequence that acts as an antigen site in an amino acid sequence essential for maintaining its enzymatic activity in a protease derived from hepatitis C virus. It forms specific binding with antibodies to hepatitis virus.

[0002]

2. Description of the Related Art Hepatitis C is known as hepatitis C virus (HC).
V; hepatitis C virus),
The route of infection is known to be blood-borne parenteral infection. Most are due to transfusion of blood contaminated with HCV, treatment with blood products made from contaminated blood, and misuse of contaminated medical devices, and mother-to-child transmission and marital transmission are extremely rare. In particular, in hepatitis C,
Many adult primary infections become chronic and become persistent. Specifically, there are statistics that about 70% of post-transfusion hepatitis C cases and about 60% of sporadic hepatitis C cases become chronic. Therefore, accurate and rapid diagnosis is important, and especially, HCV
The development of antibody testing methods has been energetically advanced.

An antibody against hepatitis C virus is HCV.
The antibody specifically reacts with either the virus particle itself or a protein translated from the HCV virus gene, and an antibody test can be performed by utilizing the specific antigen-antibody reaction. The virus particle itself cannot be used for such an antibody test, and a protein translated from the HCV virus gene or a fragment thereof is used as an antigen peptide. As shown in FIG.
There is a single open reading frame (ORF) encoding a polyprotein consisting of 000 amino acids,
The polyprotein translated from this is cleaved by the host-derived signalase and the HCV-derived protease into individual proteins.

[0004] The first used as an antigenic peptide for antibody testing is a group of various enzyme proteins required for virus replication, that is, of non-structural proteins, C4 corresponding to NS4 protein.
An antigen peptide called 100-3. Then this NS4
A polypeptide that combines the protein and the C-terminal region of the NS3 protein that precedes it, for example, an antigen peptide called C200, and further corresponds to a core protein that is a structural protein that forms a virus particle, for example, C22 An antigenic peptide referred to as was used. In addition, the use of the NS5 protein as an antigenic peptide has been achieved. In addition, it has been confirmed that these regions (FIG. 2) used for the antibody test have a somewhat high conservation in gene comparison between HCV mutants. Specifically, it has been reported that the core region is most conserved, followed by NS3, NS4, and NS5 in that order (Pharmaceutical Journal, Vol. 31, No. 8, 1987-1992 (1995) and the like). reference).

[0005] In studies of the immune response to HCV infection, in many cases where acute helper cell infection shows a helper T cell response to the NS3 protein antigen of HCV in the early stage of onset, HCV is eliminated. It has been reported that HCV cannot be eliminated in cases without this helper T cell response (HM Diepolderet).
al, Lancet., 346, 1006-1007 (1995)). In addition, regarding the involvement of the NS3 protein in the development of liver cancer caused by HCV, about 5 'half of the cDNA encoding the NS3 protein (NS3-5'; equivalent to the protease active region)
When the recombinant cells introduced into the above were planted in nude mice, 6
After a week, tumor formation was observed at a frequency of 10/10 (100%), whereas about 3 'side (NS3-3'; ATP
NIH3T3) (equivalent to the helicase active region with ase activity)
It has been reported that the frequency of tumor formation was 1/5 (20%) when the recombinant cells introduced into E. coli were planted (D. S.
akamura et al., J. Virol. 69, 3893-3896 (1995)).

[0006] As described above, not only is it an essential enzyme protein in HCV viral replication,
It has been speculated that the NS3 protein of CV, particularly the protease active region present in the N-terminal half thereof, plays a major role in the persistent infection and chronicity of hepatitis C and the accompanying development of liver cancer. Should be. In other words, in the early stage of the onset of hepatitis C, this HCV
Examination of the presence or absence of an antibody against the protease active region (HCV-derived protease) in the NS3 protein is considered to be useful in predicting the subsequent transition of the disease state. However, to date, no antibody test agent suitable for testing antibodies against this HCV-derived protease has been developed. Specifically, the epitope (amino acid sequence of the antigenic site) for an antibody against a protease derived from HCV has not been determined.

[0007]

SUMMARY OF THE INVENTION The present invention specifies an epitope sequence for an antibody against an HCV-derived protease, and provides an artificial antigen peptide containing the identified epitope sequence. It is intended to provide an antibody test agent containing a peptide. In particular, it is an object of the present invention to specify an epitope sequence in the vicinity of an enzyme active site of an HCV-derived protease, and to provide an artificial antigen peptide containing the specified epitope sequence.

[0008]

Means for Solving the Problems In order to solve the above problems, the present inventors, in the course of intensive research, first identify a partial amino acid sequence essential for the expression of the enzymatic activity of an HCV-derived protease ( Japanese Patent Application Laid-Open No. 9-9961), and then confirmed that an antibody that specifically reacts with an HCV-derived protease could be present.
The antibody binds to a protease derived from HCV,
It has been found that the enzyme activity is strongly inhibited (Japanese Unexamined Patent Publication No.
-2007676). Based on such knowledge, H
Further studies were carried out to verify whether the amino acid sequence serving as an epitope for the antibody exists in the partial amino acid sequence essential for the expression of the enzymatic activity of the CV-derived protease, and to identify the epitope sequence. As a result, at least three amino acid sequences serving as epitopes for the antibody are present in the partial amino acid sequence essential for the expression of the enzymatic activity of the HCV-derived protease, and the epitope sequence can be identified. I came to.

That is, the present invention provides the following amino acid sequence (I): Gly Trp Pro, amino acid sequence (II): Arg Arg Arg Gly and amino acid sequence (III): Asp Xaa Asp Leu Val (Xaa
Represents any natural amino acid), the amino acid residue 12 comprising at least one amino acid sequence selected from the group consisting of
The above is an antigenic peptide (chain peptide) derived from the hepatitis C virus.

The present invention further provides the following amino acid sequence (IV) containing both the amino acid sequence (I) and the amino acid sequence (III): Asp Xaa Asp Leu Val Gly
Antigenic peptide (chain peptide) of 12 or more amino acids including Trp Pro (Xaa represents any natural amino acid)
It is. Furthermore, the present invention comprises the above-mentioned chain peptide,
It is a test agent for antibodies against HCV-derived protease.

[0011]

BEST MODE FOR CARRYING OUT THE INVENTION The above partial amino acid sequence which characterizes the antigenic peptide of the present invention is a partial amino acid sequence present in the amino acid residue region of the NS3 protein of HCV, ie, a protein called p70. (Fig. 1). In particular, the peptide of the present invention comprises a protease active region existing in the N-terminal half region of the amino acid sequence of p70, specifically a region called Cpro-2 (a series of amino acid sequences encoded on the HCV genome). Of which, 190 amino acid residues from the alanine at the 1027th position to the proline at the 1185th position)
An artificial peptide containing an amino acid sequence serving as an epitope present therein.

[0012] p70 is encoded on the HCV genome
Of a series of amino acid sequences consisting of 3010 amino acids,
It is a protein consisting of 631 amino acid residues from alanine at the 1027th position to threonine at the 1657th position. The amino acid sequence and the base sequence encoding it are described in SEQ ID NO: 1 for reference.

[0013] Here, since the alanine is the 1027 th amino acids of the amino acid sequence encoded on HCV genome is the first amino acid if, based on the amino acid sequence of p70, this amino acid "Ala ¹ And the position of the amino acid at p70 is displayed and described at the upper right of the amino acid. When describing the amino acid sequence of the Cpro-2 based on this method, the amino acid sequence (I)
Corresponds to Gly ⁸⁴ -Trp ⁸⁵ -Pro ^{86 and} has an amino acid sequence (II)
Corresponds to Arg ¹¹⁷ -Arg ¹¹⁸ -Arg ¹¹⁹ -Gly ¹²⁰ (SEQ ID NO: 2), and the amino acid sequence (III) is Asp ⁷⁹ -Gln ⁸⁰ -Asp ⁸¹ -Leu ⁸²
This corresponds to -Val ⁸³ (SEQ ID NO: 3).

As elucidated earlier by the present inventors, the HC
V derived serine protease; Cpro-2 integral part amino acid sequence for enzyme activity expression are part of the Val ³⁵ ~Val ^172, in particular, sequences characteristic of chymotrypsin-like serine proteases; His ⁵⁷ ~Gly ¹⁵² (H −X ₂₂ −VXXD−X ₅₅ −GX
The SGXP-X ₉ -G) in the amino acid sequence three types serving as these epitopes are present. Incidentally, including Asp ^81, which is one of the major amino acids constituting the catalytic active site of the serine protease, epitope sequence Asp ⁷⁹ -Gln ⁸⁰ -Asp ⁸¹ -Leu
^The antibody that binds to ^82- Val ⁸³ (SEQ ID NO: 3) is considered to inhibit the enzymatic activity because it directly covers the catalytically active site. Further, the epitope sequence Gly adjacent to Asp ⁸¹ described above.
Antibodies that bind to the ⁸⁴ -Trp ⁸⁵ -Pro ^86, to the process of the substrate to the catalytic active site is inserted, is believed to inhibit the enzyme activity since steric hindrance.

The three types of amino acid sequences serving as the above epitopes can be specified as follows. That is, as already proposed by the present inventors, there are a plurality of mouse monoclonal antibodies that bind to the Cpro-2 and inhibit the enzyme activity thereof (see Japanese Patent Application Laid-Open No. 9-206076). The three amino acid sequences described above are specified as amino acid sequences that bind to this monoclonal antibody. Specifically, the presence of the above three epitope sequences is identified from the amino acid sequence of the antigen peptide to which the IgG antibody produced by the three hybridoma cell lines shown in Table 1 below binds. In addition, these hybridoma cell lines, JE-7E3 (FERM B
P-5279), JE-7E9 (FERM BP-5280), JE-8D4 (FER
MBP-5281) have all been deposited with the National Institute of Bioscience and Human-Technology (National Institute of Advanced Industrial Science and Technology) under the identification number and the accession number assigned by the depositary institution.

[0016]

[Table 1]

In the present invention, the above-mentioned hybridoma cell lines JE-7E3 (FERM BP-5279) and JE-7E9 (FERM BP-5279) are used.
BP-5280), Cpr produced by JE-8D4 (FERM BP-5281)
IgG antibodies against o-2 were mAb 7E3, mAb 7E9,
Called mAb 8D4.

In general, a protein translated in a host cell, which is not normally secreted outside the cell,
There is no immunity to the intracellular protein, and therefore no antibody to it. However, these non-structural proteins derived from HCV are extracellular when the host cell is killed or damaged due to new production of the virus in the host cell, so that immunity against these non-self proteins occurs. Actually, a human antibody against the NS4 protein, that is, an antibody detected by the C100-3 antigen peptide is known. Considering this point, it has not been reported so far, but when humans suffer from HCV, antibodies to NS3 protein, especially antibodies to Cpro-2, as well as antibodies to NS4 protein are produced. Inferred.

The epitope on the antigen peptide that reacts with each of the antibodies mAb 7E3, mAb 7E9 and mAb 8D4 was determined as follows. As a pseudo-antigen peptide, a commercially available random peptide library (trade name: FliTRX Rando)
m Peptide Display Library (Invitrogen)) to select multiple types of amino acid sequences that can bind to each antibody,
The partial amino acid sequence information common to the amino acid sequences of these selected pseudo antigenic peptides was extracted.

The random peptide library contains 12
A modified library consisting of a random library consisting of 10 polypeptides inserted into the active site of E. coli-derived Thioredoxin protein was added to Flagellin, which constitutes E. coli flagella.
Nonessential domain in proteins
It was presented on the flagella as a fusion protein inserted into it. In this fusion protein, a random library composed of 12 polypeptides does not interact with other domains constituting the fusion protein itself, but interacts only with another protein to form an intermolecular bond. It takes a form that can be done. In particular, the library is in a form suitable for binding to an antibody and is designed for use in determining the epitope of an antibody (Z. Lu et al., Bio / Technology Vol. 13, 366). −372
(1995)). The repertoire of random parts is 1.77
× 10 ⁸ types, and there are many types of peptide chains that react with the antibody. Thus, by selecting a peptide that reacts with the monoclonal antibody from among these many peptide chains, and comparing the amino acid sequences, a partial amino acid sequence essential for binding to the antibody can be extracted.

The random peptide library is a recombinant bacterial library in which the above-mentioned fusion protein has been incorporated into a known plasmid and introduced into host Escherichia coli. The target antibody is adsorbed on a plate. ,
A culture of a recombinant bacterium of the library is added to the surface, and the remaining Escherichia coli is washed away, leaving the bacterium fixed by the binding between the antigen peptide and the antibody displayed on the flagella. Thereafter, the fixed recombinant bacteria are recovered by cutting the flagella by mechanical means such as ultrasonic cleaning. The recovered recombinant bacteria are cultured again to obtain a collection of the selected recombinant bacteria.

Further, the same operation is repeated several times, usually four more times, using this assembly, to produce an antigen peptide having a binding ability with a target antibody of a certain level or more as a fusion protein. Only bacteria can be collected. The bacterial population selected by this clone selection (panning) operation is isolated as a strain that forms a single colony by seeding and culturing on an agar medium plate according to a conventional method. Each clonal strain is further cultured separately, a plasmid introduced therein is collected, and a nucleotide sequence encoding a peptide chain incorporated in the plasmid is analyzed by Sanger method or the like. In this peptide library, the base sequence encoding the peptide chain to be analyzed is the base sequence of the partial DNA shown in FIG. 3, and the base sequence shown in FIG. Analysis can be performed.

Forward primer: 5'-ATTCACCTGACTG
ACGAC-3 '(SEQ ID NO: 5) Reverse primer: 5'-CCCTGATATTCGTCAGCG-3' (SEQ ID NO: 6) The nucleotide sequence encoding a peptide chain capable of binding to each of the obtained antibodies was replaced with the corresponding amino acid sequence. Is compared with the amino acid sequence of Cpro-2, and a partial sequence having high similarity is selected. Usually, in the above-mentioned clone selection (panning) operation, a plurality of species are selected, and each expresses a peptide chain having a slightly different amino acid sequence. As those that satisfy homology with these multiple amino acid sequences, Cpro
A specific partial sequence in the -2 amino acid sequence is selected. That is, for each antibody, the partial amino acid sequence serving as its antigen is determined.

Specifically, the above mAb 7E3, mAb 7E9,
b For each antibody of 8D4, a peptide chain shown in FIGS. 5 to 7 (SEQ ID NOs: 7 to 43) by a clone selection (panning) operation
Are selected as having binding ability.

From the comparison, in the case of mAb 7E3, the amino acid sequence: a peptide site corresponding to Gly ⁸⁴ -Trp ⁸⁵ -Pro ⁸⁶ , and in the case of mAb 7E9, the amino acid sequence: Arg ¹¹⁷ -Arg ¹¹⁸ -A
a peptide site corresponding to rg ¹¹⁹ -Gly ¹²⁰ (SEQ ID NO: 2) and the amino acid sequence for mAb 8D4: Asp ⁷⁹ -Gln ⁸⁰ -Asp
It is found that each binds to a peptide site corresponding to ^81- Leu ⁸² -Val ⁸³ (SEQ ID NO: 3). Therefore, these
It is considered that human antibodies having the same antigen specificity as mAb 7E3, mAb 7E9, and mAb 8D4 are also present in patients with hepatitis C virus. For detection of these human antibodies, the amino acid sequences (I) to (III) ) Can be used as the antigen peptide. In addition, since these antigenic peptides have the amino acid sequences (I) to (III) derived from the hepatitis C virus at sites directly involved in binding to the antibody, high reactivity and selectivity are achieved. it can.

[0026] Additionally, comprising the amino acid sequence (I) and amino acid sequence (III) together, at least 12 amino acid residues or more linear peptide comprising the amino acid sequence (IV), i.e., Asp ⁷⁹ -Gln ⁸⁰ -Asp ⁸¹ - Leu ⁸² -Val ⁸³ -Gly ⁸⁴ -Trp ⁸⁵ -Pro
^The peptide site corresponding to ⁸⁶ (SEQ ID NO: 4) was mAb 7E3
It is more preferable because it can bind to any of human antibodies corresponding to mAb 8D4. In the amino acid sequence (III) or the amino acid sequence (IV), the second amino acid represented by Xaa is an amino acid found in nature (for example, 20 kinds of amino acids), and Gln, Arg, Leu, Ala, Se
r, Val, Asn can be preferably used, and Gln, Arg, L
eu, Ala and Ser are more preferred, and the original Gln is even more preferred.

On the other hand, the amino acid sequence of the peptide portion extended to both ends of the amino acid sequences (I) to (III) essential for the antigenic peptide of the present invention is not only the amino acid sequence of the hepatitis C virus itself, but also the amino acid sequence of FIG. As shown in Nos. To 7, even a peptide in which some amino acid sequences are mutated such as deletion, substitution, or addition can maintain the binding ability to an antibody. Further, regarding the amino acid sequence of the peptide portion extended to both ends of the amino acid sequence (IV), the N-terminal side of the amino acid sequence (III) shown in FIG. The amino acid sequence at the C-terminal side of the amino acid sequence (I) to be used can be arranged. In FIG. 5, italics indicate an amino acid sequence outside the random region.

The chain peptide containing any one of these amino acid sequences (I) to (III) or (IV) has at least 12 amino acid residues and generally has 5 or more amino acid residues.
It is preferably 0 amino acid residues or less, preferably 40 amino acid residues or less, and particularly preferably about 30 to 40 amino acid residues. That is, the amino acid sequences (I) to (II) conferring reactivity with the antibody
The sequence of I) or amino acid sequence (IV) is arranged at the center, and approximately 1
Assuming that the peptide chains of 0 to 15 amino acid residues are respectively extended, the surplus partial peptide chains play a suitable role in immobilizing the antigen peptide on a support, for example, a plate. The amino acid sequence of the peptide portion extending to both ends of the essential portion consisting of the 12 amino acid residues may be appropriately selected so as to be useful for immobilization on a support. Specifically, an amino acid sequence other than the epitope sequence can be diverted in an antigen peptide conventionally used for antibody testing.
Alternatively, in the amino acid sequence of Cpro-2, an amino acid sequence adjacent to a site corresponding to amino acid sequence (I) to (III) or amino acid sequence (IV) may be used as it is.

Since the antigenic peptide of the present invention generally has 50 amino acid residues or less, particularly about 30 to 40 amino acid residues, it may be chemically synthesized by applying a known solid-phase peptide synthesis method. These synthesized peptides are
It can be purified by reversed-phase liquid chromatography or the like. In addition, when the amino acid sequence (I) to (III) or the amino acid sequence (IV) is arranged at the center of the peptide fragment, by-products having a slight difference in peptide chain length can also react with the antigen peptide. Since there is no difference in sex, a partially purified peptide may be used in addition to a purely purified peptide.

The antibody test agent containing the antigenic peptide of the present invention is usually used as a diagnostic agent for serodiagnosis. In this case, various diagnostic agents can be designed depending on the immunological detection method employed. it can. When a one-way plate immunodiffusion method, an immunoelectrophoresis method, an immunoadhesion hemagglutination method, a complement fixation method, or the like is used as a detection method, the antigen peptide of the present invention is used as it is, and is commonly used in individual test methods. It may be prepared in the form of a test drug.

As an example, in the one-way plate immunodiffusion method, a support such as agar, agarose or polyacrylamide is appropriately selected, dissolved in a buffer, and a predetermined amount of an antigen peptide is added and mixed. Is flowed flat into a glass plate or a plastic container and solidified to prepare a solidified gel plate. Thereafter, a hole for injecting the test serum is appropriately formed and used.

When applied to the hemagglutination method, an antigen peptide is bound to fine particles serving as nuclei. As the fine particles, blood cells of mammals or birds are used. In addition, particles having a diameter of several μm such as polystyrene latex, polyester latex, bentonite, and glass beads may be used. For binding to the particles, a suitable one may be selected from reagents such as glutaraldehyde, formaldehyde, tannic acid, and bisdiadizebenzidine, depending on the particles.

When used for radioimmunoassay (RIA), enzyme-linked immunosorbent assay (ELISA), etc., an antigen peptide is bound to an appropriate solid phase surface. As the solid phase, commonly used microplates, tubes, beads, magnetic beads and the like can be used.

The antibody bound to the antigenic peptide of the present invention is detected by a conventional means according to each assay method, and the test agent is prepared as a kit together with these conventional detection reagents. Can be. Further, it may be used in combination with another hepatitis C virus antibody testing antigen as a combined test drug. In addition, the amino acid sequences (I) to
Two or more antigenic peptides containing any of (III) may be used in combination. In other words, only the antigen peptide is different, and the preparation method and the like, such as the reagents to be included in the test agent and the like, may be similar to those of conventional antibody test agents of this type.

[0035]

EXAMPLES Hereinafter, the antigenic peptide of the present invention and its reactivity will be described more specifically with reference to examples. However,
The technical scope of the present invention is not limited to these Examples.

(Example 1) The inventors have already determined that HCV
It has been confirmed that there is an antibody that binds to the protease Cpro-2 of interest and inhibits its enzyme activity (Japanese Patent Application Laid-Open No.
No. 206076). Specifically, hybridoma cell lines, JE-7E3 (FERM BP-5279), JE-7E9 (FERM BP
-5280), Cpro produced by JE-8D4 (FERM BP-5281)
IgG antibodies against -2 (mAb 7E3, mAb 7E9,
Ab 8D4) was presumed to have an amino acid sequence serving as an epitope for the antibody in the partial amino acid sequence essential for the expression of the enzyme activity of Cpro-2.

For the purpose of specifying the epitope sequences of these three types of antibodies, a commercially available random peptide library (trade name: FliTRX Random Peptide Display Library (Intradeal), in which 12 consecutive amino acids have a random sequence)
Utilizing in vitrogen)), the amino acid sequences of peptide chains capable of binding to each antibody were selected. The random peptide library is obtained by converting a random library composed of 12 polypeptides into Thioredoxin derived from E. coli.
The modified protein inserted into the active site of the protein is further displayed on the flagella as a fusion protein inserted into the nonessential domain of the Flagellin protein constituting the flagella of Escherichia coli ( (Figure 11). In this fusion protein, a random library composed of 12 polypeptides does not interact with other domains constituting the fusion protein itself, but only interacts with another protein to form an intermolecular bond. It takes a form that can be done. In particular, the random library is in a form suitable for binding to an antibody, and is designed to be used for determining the epitope of an antibody (Z. Lu et a).
l., Bio / Technology Vol. 13, 366-372 (1995)).

The hybridoma cell line JE-7E3 (FE
RM BP-5279), JE-7E9 (FERM BP-5280), JE-8D4
(FERM BP-5281) produced IgG antibodies against Cpro-2 (mAb 7E3, mAb 7E9, and mAb 8D4, respectively) obtained by culturing each hybridoma cell line according to a conventional method and isolating from the culture. (See JP-A-9-206076).

Selection of strains expressing a peptide that binds to each mouse monoclonal antibody, and determination of the amino acid sequence of the peptide chain were performed using the instructions and literature attached to the kit for the random peptide library (Z. Lu et a).
l., Bio / Technology Vol. 13, 366-372 (1995)) and performed according to the standard procedure. The outline of the procedure actually used is described below.

Operation Procedure 1. Preparation of Culture Solution Various culture solutions and the like used in the following operations were prepared to the compositions shown in Tables 2 to 4 below according to the instructions attached to the kit.

10 × M9 salt solution (Table 2)

[0042]

[Table 2]

IMC medium (Table 3)

[0044]

[Table 3]

RM medium (Table 4)

[0046]

[Table 4]

2. Cultivation of FliTrx library (inoculum) The library (inoculum) attached to the kit was subcultured in 50 ml of IMC medium, and cultured overnight (about 15 hours) with stirring at 25 ° C. Take a very small amount of sample from the culture and
The absorbance OD ₆₀₀ at 0 nm was measured, and from the value, 1 OD ₆₀₀ =
Approximately 1 × 10 ⁹ cells (cells) were converted to estimate the number of E. coli.
Take a culture solution (usually 2-3 ml) containing 1 × 10 ¹⁰ Escherichia coli and add 50 μm IMC medium containing 100 μg / ml tryptophan.
After dilution to 1 liter, the mixture was cultured at 25 ° C. for 6 hours with stirring to express the peptide library in the flagella of this Escherichia coli.

3. Selection of clones binding to antibody (panning operation) The following two types of techniques were used for panning. (1) Panning using a plate An aqueous solution (1 ml) containing 20 μg of an antibody was added to a plate (Falcon 3002) having a diameter of 60 mm, and the plate was allowed to stand at room temperature for 1 hour to allow the antibody to be adsorbed on the plate surface. Remove the aqueous solution containing the antibody,
Rinse with 5 ml sterile water, then 7 ml blocking solution
(IMC medium, 1% non-fat dry milk, 150 mM NaCl, 0.4% α-methyl mannoside, 100 μg / ml ampicillin), gently stirred, and left at room temperature for 1 hour.

Next, 0.5 g of non-fat dry milk, 1.5 ml of 5M NaCl, and 2.5 ml of sterilized 20% α were added to 50 ml of an E. coli culture cultured for 6 hours in the above-described culture solution containing tryptophan.
After addition of -methyl mannoside, 7 ml of this solution was added to the plate on which the antibody was immobilized. The mixture was gently stirred, allowed to stand at room temperature for one hour, and then the E. coli culture was discarded. To remove E. coli that has not bound to the antibody adsorbed on the plate surface, remove the plate from 7 ml of the washing solution (IMC medium, 0.4% α-
(Methyl mannoside) three times.

Finally, after removing the washing solution (while leaving a small amount of solution), in order to recover the E. coli remaining on the plate, vigorously vortex (ultrasonic washing) in order to tear off the flagella bound to the antibody. Was applied. Then 10 m
1 l of IMC medium was added, the detached Escherichia coli was recovered, transferred to a flask, and cultured at 25 ° C overnight.

From the obtained culture of Escherichia coli subjected to the primary selection, a culture (usually 2-3 ml) containing 1 × 10 ¹⁰ Escherichia coli was again taken, and the IMC medium containing 100 μg / ml tryptophan was collected. After dilution to ml, the mixture was cultured at 25 ° C. for 6 hours with stirring to express a peptide library in the flagella of Escherichia coli. A series of operations (panning) described above was performed to perform a second sorting.

Further, the same operation was performed three times, and a total of five stages of selection were repeated, and a plurality of Escherichia coli subjected to the fifth selection having high binding ability to antibodies was obtained. In addition, the isolation | separation of each strain clone used the technique of "4. isolation | separation of a positive clone" mentioned below.

(2) Panning Using Magnetic Beads (Magnetic Cell Sorter) 8 ml of Escherichia coli cultured for 6 hours in the above-mentioned culture solution containing tryptophan was taken into a microtube, and centrifuged (4,000 rpm, 3 min) to collect the Escherichia coli. 0.8 ml wash buffer (IMC medium, 150 mM
NaCl, 0.2mg / ml BSA, 0.4% α-methyl mannoside)
Washing was repeated and finally suspended in 0.5 ml of a washing buffer. After adding 20 μg of a mouse monoclonal antibody, the mixture was gently stirred with a pipette, and allowed to stand at room temperature for 30 minutes. Escherichia coli was recovered by centrifugation, and the suspension in washing buffer (0.8 ml) was repeated twice to remove excess antibody. Thereafter, the recovered E. coli was suspended in 160 ml of a washing buffer.

Next, Escherichia coli bound to the target antibody was separated from the recovered Escherichia coli by a magnetic cell separation method (S. Miltenyi
et al., Cytometry 11, 231-238 (1990)). The magnetic cell separation system is Milt
The one from enyi Biotec GmbH (Bergisch Gladbach, Germany) was used.

Goat-anti-mouse immunoglobulin G (H + L) F
Magnetic beads with (ab ') 2 covalently attached (Indirect
Microbeads Goat-anti-mouse-IgG, Miltenyi Biotec, G
(ermany) 40 ml and gently stirred. In the refrigerator (about 8
(C) for 15 minutes, centrifuged to collect E. coli, remove extra beads, and suspended in 0.5 ml of washing buffer. This E. coli suspension was set in a strong magnetic field and 3 ml
Separation column (Separation
Column, type LS +), and washed three times with 3 ml of a washing buffer to wash out Escherichia coli to which no beads were bound. After removing the separation column from the magnetic field, 5 ml of IMC
Add the medium and sterilize the desired E. coli
Collected during l). The culture solution was adjusted to 10 ml by adding 5 ml of IMC medium, and cultured at 25 ° C. overnight.

From the obtained culture solution of E. coli subjected to the primary selection, a culture solution containing 1 × 10 ¹⁰ Escherichia coli (usually 2-3 ml) was again taken, and the IMC medium containing 100 μg / ml tryptophan was collected. After dilution to ml, the mixture was cultured at 25 ° C. for 6 hours with stirring to express a peptide library in the flagella of Escherichia coli. A series of operations (panning) described above was performed to perform a second sorting. In the second panning operation, the initial amount of E. coli and the amount of the target antibody were halved.

As a result of the second selection, a plurality of Escherichia coli having high antibody-binding ability were obtained. In addition, the isolation | separation of each strain clone used the technique of "4. isolation | separation of a positive clone" mentioned below.

4. Isolation of positive clones
After spreading on M medium and culturing at 30 ° C. overnight (16 to 24 hours), a single colony was picked, planted in 2 ml of RM medium, and then cultured for 30 minutes.
Stirred at 16 ° C for 16 to 24 hours. 0.1 ml of this E. coli
After dilution in 5 ml of IMC medium containing 00 μg / ml tryptophan, the mixture was stirred and cultured at 37 ° C. for 8 or 16 hours to express a peptide library. After adding overnight glycerol to a final concentration of 20% for the remaining E. coli cultured overnight,
Saved in.

5. Confirmation of Positive Clones by Western Blotting The clones obtained by this separation were expressed in the above peptide library, and then confirmed to have a peptide chain that actually binds to the target antibody. That is, the peptide library should have been inserted into thioredoxin fused to a protein (furandulin) constituting flagella of Escherichia coli. Therefore,
Using both the mouse antibody against thioredoxin itself and the desired antibody, the furundulin protein into which the peptide chain binding to the desired antibody was inserted was confirmed by Western blotting.

4 ml of the culture solution of Escherichia coli expressing the peptide library was collected, and immediately 50 ml of SDS-sample buffer (0.125 M Tris-HCl, pH 6.8, 20% glycerol, 0.4% SDS, 0.01% bromo (Phenol blue) was added and suspended. The viscosity of the solution was reduced by sonication to break the high molecular weight DNA, and the mixture was incubated at 95 ° C for 3 minutes. Centrifuge (15,000 rpm, 10 min) to remove insolubles, and use SDS-PAGE
For use as samples. After electrophoresis on a 12.5% polyacrylamide gel, blotting was performed on a commercial PVDF membrane (Trans-Blot transfer medium 0.2 mm, Bio-Rad Laboratories).

By performing the above treatment, the binding between the antigen peptide and the target antibody may be weakened, or the binding between the thioredoxin protein into which the antigen peptide is inserted and the antibody against thioredoxin itself may be weakened. As expected, binding to these primary antibodies was performed overnight at 4 ° C. A commercially available reagent was used for band detection in Western blotting, and the labeling enzyme method was applied.
Secondary antibody was biotinylated goat anti-mouse IgG (Life Tec
h.), a streptavidin-peroxidase conjugate (Tago, Inc.) was bound to this biotinylated secondary antibody as a labeling enzyme, and then DAB Peroxidase Su
Bands were detected using bstrate Tablet Set (Sigma) as a chromogenic substrate.

In the positive clone in which strong binding was confirmed with both the mouse antibody to thioredoxin itself and the desired antibody, the 12-amino acid peptide chain portion inserted in this peptide library was replaced with the desired antibody. It was confirmed that an epitope capable of binding was present (FIG. 8). In FIG. 8, the results of electrophoresis of a lysate of an E. coli clone (clone # 35, 38, 53, 121) that binds to mAb8D4 are shown in lanes 1, 2, 3, and 4, respectively. The result of the electrophoresis is shown in lane 5 (FIG. 8).
(A)).

The obtained band was blotted on a PVDF membrane, and the result of Western blotting using an anti-thioredoxin monoclonal antibody as a probe is shown in FIG. 8 (B).
FIG. 8 (C) shows the results obtained by using the monoclonal antibody 8D4 as a probe. The samples in each lane are the same as described above.

6. Identification of Amino Acid Sequence of Antigenic Peptide Chain of Positive Clone For the positive clone confirmed by the above-described Western blotting method, the inserted peptide chain portion of 12 amino acid residues and a region adjacent thereto, that is, as shown in FIG. The amino acid sequence encoded by the recombinant gene region was identified by the following procedure.

The peptide library used was recombined using the plasmid pFliTrx (Invitrogen) as shown in FIG. Therefore, the plasmid pFliTrx was collected from each clone, and the nucleotide sequence of the random peptide coding region was identified in the known nucleotide sequence shown in FIG. 3 and identified as the amino acid sequence encoded thereby.

After the above positive clone confirmation is completed,
Escherichia coli cultured overnight is glycerol (final concentration 20%)
And stored at -70 ° C. A part of the E. coli was spread on an RM agar (RM medium, 1.5% Bacto Agar) plate and cultured at 30 ° C. for 20 to 30 hours to isolate a single colony. Plasmids were prepared by picking 30 colonies. Alternatively, if the yield of the plasmid is very poor, E. coli cultured in 25 ml of RM medium can be prepared using a commercially available kit (Qu
By preparing the plasmid using iagen tip-20), a plasmid capable of DNA sequencing was obtained.

When [α- ³² P] dCTP was used, DNA sequencing was performed using the Sequenase version 2 DNA Sequencing Kit (A
mersham) and AutoCycle for automated DNA sequencers.
DNASequencing Kit (Pharmacia) and a primer labeled FITC at the 5 'end were used. In both methods, the primers used were FliTrx (5'-ATG TAC TCA AAG CGG specified in this random peptide library kit).
ACG GGG CGA-3 ') (SEQ ID NO: 44) and RSR (5'-TTG CCC
TGA TAT TCG TCA GCG-3 ') (SEQ ID NO: 45).

The base sequence of the DNA was determined for both the positive and negative strands,
It was confirmed that the results agreed. The two primers correspond to the positions shown in FIG. By comparing a known nucleotide sequence in this region with a nucleotide sequence obtained as a result of DNA sequencing, a nucleotide sequence encoding an amino acid of the expressed peptide chain was derived. That is,
By superimposing the nucleotide sequence shown in FIG. 3 and the nucleotide sequence elucidated in each clone, the 36 bp nucleotide sequence encoding the peptide can be identified, and the amino acid sequence translated from the 36 bp nucleotide sequence is determined. .

In this example, the panning method using a plate was applied to the mAb 7E9 antibody and the mAb 8D4 antibody,
For b7E3 antibody, a panning method using magnetic beads (magnetic cell sorter) was applied. For each of the positive clones selected by the above procedure for each antibody, the inserted peptide chain portion of 12 amino acid residues and the amino acid sequence of the region adjacent thereto were identified.

One example of the results is shown in FIGS. When the homologous portions in the peptide chain portion of 12 amino acid residues binding to each antibody were made to correspond to each other, it was found that the partial sequences surrounded by a frame in FIGS. 5 to 7 had high homology. These mAb 7E9 antibodies, mAb 8D4
Both the antibody and the mAb 7E3 antibody are prepared using the fusion type Cpro-2 protein as an immunizing antigen and bind to Cpro-2, and thus correspond to the highly homologous partial sequences shown in FIGS. The amino acid sequence should be found in the amino acid sequence of Cpro-2. As expected, sequences corresponding to the highly homologous partial sequences of these positive clones were found in the amino acid sequence of Cpro-2 (FIG. 5).
7).

That is, for the mAb 7E3 antibody, Gly ⁸⁴ -Trp
^85- Pro ⁸⁶ , against mAb 7E9 antibody, Arg ¹¹⁷ -Arg ¹¹⁸ -A
rg ¹¹⁹ -Gly ¹²⁰ , Asp ⁷⁹ -Gln ⁸⁰ for mAb 8D4 antibody
-Asp ⁸¹ -Leu ⁸² -Val ⁸³ corresponded, and it was concluded that these were included in the epitope of each antibody.

In addition, as shown in FIGS.
Even a sequence slightly different from the partial sequence in the amino acid sequence of -2 has binding ability to the used antibody. However, the binding ability differs depending on which amino acid is different. For example, Asp ^79- as shown in the Western blotting results for mAb 8D4 antibody (FIG. 8).
In comparison of three partial amino acid sequences similar to Gln ⁸⁰ -Asp ⁸¹ -Leu ⁸² -Val ⁸³ , clone # 35 (lane 1) and clone # 121 containing the sequence of Asp-Xaa-Asp-Leu-Val were compared.
(Lane 4) shows high binding ability, but the amino acid sequence is As
Clone # 38 which differs from the sequence of p-Gln-Asp-Leu-Val by two
(Lane 2) and clone # 53 (Lane 3) are presumed to have slightly poor binding ability.

As a result of such comparison of the binding ability, mAb 7E3 was required to maintain a high binding ability for each antibody.
For antibodies, Gly ⁸⁴ -Trp ⁸⁵ -Pro ⁸⁶ the same amino acid sequence, for the mAb hybridoma selected from 7E9 antibody, Arg ¹¹⁷ -Arg ¹¹⁸ -Arg ¹¹⁹ -
The same amino acid sequence as Gly ¹²⁰ (SEQ ID NO: 2) is mAb 8D4
For ^{antibodies, Asp 79 -Gln 80 -Asp 81 -Leu} 82 -Val 83 identical or similar to Asp-Xaa-Asp-Leu- Val (SEQ ID NO: 4
6) turned out to be preferred.

The partial sequence serving as these epitopes is C
Regarding the position of the protease Cpro-2 molecule derived from hepatitis C, the protease Cpro-2 of the HCV strain BK strain which is a different hepatitis C virus strain was determined.
Crystal structure analysis results of two molecules (RA Love et al., Cell
87, 331-342 (1996)), and the three-dimensional position is illustrated.

FIG. 9 shows the result. FIG. 9 shows the active site of the protein, including three residues (His ⁵⁷ , Asp ⁸¹ , Ser ¹³⁹ ) involved in catalysis, indicated by amino acids represented by one letter. The portion forming the secondary structure, specifically, the β chain, α helix, and the β chain (A1-F1) in the trypsin-like barrel structure are indicated by a ribbon. Three amino acid residues at the catalytic center (His ⁵⁷ , Asp
⁸¹ , Ser ¹³⁹ ) is shown by its side chain structure. In FIG. 9, specifically, the epitope positions of the mAb 7E3 antibody and the mAb 8D4 antibody correspond to Asp located at the active center of this protease.
⁸¹ and Its is adjacent portions, the epitope location of the mAb7E9 antibody, rather than the active center, it can be seen that located in the protease protein surface.

FIG. 10 shows the correspondence between the amino acid sequence of HCV BK NS3P and the amino acid sequences of other serine proteases based on the superposition of the crystal structures of these proteins. The underlined portions are residues constituting the β chain. The general position of each β-chain in all sequences is indicated by a dotted arrow (---- →). The labeling of these chains is
Extra N-terminal β-chain (A0-D
The practice was followed except for 0). The three amino acid residues involved in catalysis are marked by an asterisk (*) at the head. Excess amino acid residues not shown are indicated by the number of amino acids in parentheses.

The abbreviations for proteins are as follows: SVCP: Sindbis virus core protein; ALP: α-lytic pro
teinase; ELA: porcine elastase; HRV14: human rhin
ovirus type 14 3C proteinase; HCVBK: hepatitis C v
irus, BK strain NS3 proteinase

The amino acid numbers are given for HCVBK (upper) or ELA (lower).

As shown in FIG. 10, the HCV strain BK
In the amino acid sequence of the protease Cpro-2 of the strain,
Similar to Gly ⁸⁴ -Trp ⁸⁵ -Pro ⁸⁶ for mAb 7E3 antibody
Gly-Trp-Gln, for mAb 7E9 antibody, Arg ¹¹⁷ -Arg
^The same amino acid sequence as ^118- Arg ^119- Gly ¹²⁰ exists, but the same sequence as Asp ^79- Gln ^80- Asp ^81- Leu ^82- Val ⁸³ exists for mAb 8D4 antibody.

That is, in this HCV strain BK strain, m
It is expected that there are human antibodies corresponding to Ab 7E3 antibody, mAb 7E9 antibody, and mAb 8D4 antibody. Considering the above points, mAb 7E3 antibody, mAb 7E9 antibody, mAb 8D4 antibody is HCV-2J
Strains (N. Kato et al., Proc. Natl. Acad. Sci. USA 87,
9524-9528 (1990)), but the epitopes of these antibodies are conserved across viral strains, and the antigenic peptides of the present invention have a broad spectrum of hepatitis C It turns out to be an effective test drug for virus strains.

The antigenic peptide of the present invention has an amino acid sequence having extremely high homology across types of virus strains in the hepatitis C virus-derived protease Cpro-2 molecule,
Amino acid sequence corresponding to Gly ⁸⁴ -Trp ⁸⁵ -Pro ⁸⁶ (I): Gly
-Trp-Pro, same amino acid sequence as Arg ¹¹⁷ -Arg ¹¹⁸ -Arg ¹¹⁹ -Gly ¹²⁰ (II): Arg-Arg-Arg-Gly, Asp ⁷⁹ -Gln ⁸⁰ -Asp ⁸¹ -L
eu ⁸² -Val corresponding amino acid sequence ^{83 (III): Asp-Xaa} -A
sp-Leu-Val (SEQ ID NO: 46) or Asp ⁷⁹ -Gln ⁸⁰ -Asp
Amino acid sequence corresponding to ^81- Leu ⁸² -Val ⁸³ -Gly ⁸⁴ -Trp ⁸⁵ -Pro ⁸⁶ (IV): Asp-Xaa-Asp-Leu-Val-Gly-Trp-Pro (SEQ ID NO: 47) is essential for epitope It is contained as an amino acid sequence and can be used for detection of an antibody against the protease Cpro-2 for a wide range of hepatitis C virus strains. In particular, the antigen peptide containing the amino acid sequence (III) and / or the amino acid sequence (IV) corresponds to an epitope located at the enzyme active site of the protease Cpro-2, and is used for an antibody against hepatitis C virus. It has the advantage of being highly specific. These antibody test agents utilizing the antigenic peptide of the present invention are the same as those of the conventional antigenic polypeptides c-7, c33c and the like.
In the area, different parts are used, and they complement each other, and a test drug combining these two has an effect of further improving the accuracy of the test.

[0082]

Industrial Applicability According to the present invention, there are provided an antigen peptide against an antibody against an HCV-derived protease, and an antibody test agent containing the antigen peptide.

[0083]

[Sequence list]

 SEQ ID NO: 1 Sequence length: 1893 Sequence type: nucleic acid Number of strands: double-stranded Topology: linear Sequence type: cDNA to Genomic RNA Origin Organism name: Hepatitis C virus Strain name: HCV-J sequence Characteristic of the symbol: Characteristic code: CDS Location: 1..1893 Sequence GCG CCT ATC ACG GCC TAT TCC CAA CAA ACG CGG GGC CTG CTT GGC TGT 48 Ala Pro Ile Thr Ala Tyr Ser Gln Gln Thr Arg Gly Leu Leu Gly Cys 1 5 10 15 ATC ATC ACT AGC CTC ACA GGT CGG GAC AAG AAC CAG GTC GAT GGG GAG 96 Ile Ile Thr Ser Leu Thr Gly Arg Asp Lys Asn Gln Val Asp Gly Glu 20 25 30 GTT CAG GTG CTC TCC ACC GCA ACG CAA TCT TTC CTG GCG ACC TGC GTC 144 Val Gln Val Leu Ser Thr Ala Thr Gln Ser Phe Leu Ala Thr Cys Val 35 40 45 AAT GGC GTG TGT TGG ACC GTC TAC CAT GGT GCC GGC TCG AAG ACC CTG 192 Asn Gly Val Cys Trp Thr Val Tyr His Gly Ala Gly Ser Lys Thr Leu 50 55 60 GCC GGC CCG AAG GGT CCA ATC ACC CAA ATG TAC ACC AAT GTA GAC CAG 240 Ala Gly Pro Lys Gly Pro Ile Thr Gln Met Tyr Thr Asn Val Asp Gln 65 70 75 80 GAC CTC GTC GGC TGG CCG GCG CCC CCC GGG GCG CGC TCC ATG ACA CCG 288 Asp Leu Val Gly Trp Pro Ala Pro Pro Gly Ala Arg Ser Met Thr Pro 85 90 95 TGC ACC TGC GGC AGC TCG GAC CTT TAC TTG GTC ACG AGG CAT GCC GAT 336 Cys Thr Cys Gly Ser Ser Asp Leu Tyr Leu Val Thr Arg His Ala Asp 100 105 110 GTC ATT CCG GTG CGC CGG CGA GGC GAC AGC AGG GGG AGT CTA CTC TCC 384 Val Ile Pro Val Arg Arg Arg Gly Asp Ser Arg Gly Ser Leu Leu Ser 115 120 125 CCT AGG CCC GTC TCC TAC CTG AAG GGC TCC TCG GGT GGA CCA CTG CTT 432 Pro Arg Pro Val Ser Tyr Leu Lys Gly Ser Ser Gly Gly Pro Leu Leu 130 135 140 TGC CCT TCG GGG CAC GTT GTA GGC ATC TTC CGG GCT GCT GTG TGC ACC 480 Cys Pro Ser Gly His Val Val Gly Ile Phe Arg Ala Ala Val Cys Thr 145 150 155 160 CGG GGG GTT GCG AAG GCG GTG GAC TTC ATA CCC GTT GAG TCT ATG GAA 528 Arg Gly Val Ala Lys Ala Val Asp Phe Ile Pro Val Glu Ser Met Glu 165 170 175 ACT ACC ATG CGG TCT CCG GTC TTC ACA GAC AAC TCA TCC CCT CCG GCC 576 Thr Thr Met Arg Ser Pro Val Phe Thr Asp Asn Ser Ser Pro Pro A la 180 185 190 GTA CCG CAA ACA TTC CAA GTG GCA CAT TTA CAC GCT CCC ACT GGC AGC 624 Val Pro Gln Thr Phe Gln Val Ala His Leu His Ala Pro Thr Gly Ser 195 200 205 GGC AAG AGC ACC AAA GTG CCG GCT GCA TAT GCA GCC CAA GGG TAC AAG 672 Gly Lys Ser Thr Lys Val Pro Ala Ala Tla Ala Ala Gln Gly Tyr Lys 210 215 220 GTG CTC GTC CTA AAC CCG TCC GTT GCT GCC ACA TTG GGC TTT GGA GCG 720 Val Leu Val Leu Asn Pro Ser Val Ala Ala Thr Leu Gly Phe Gly Ala 225 230 235 240 TAT ATG TCC AAG GCA CAT GGC ATC GAG CCT AAC ATC AGA ACT GGG GTA 768 Tyr Met Ser Lys Ala His Gly Ile Glu Pro Asn Ile Arg Thr Gly Val 245 250 255 AGG ACC ATC ACC ACG GGC GGC CCC ATC ACG TAC TCC ACC TAT GGC AAG 816 Arg Thr Ile Thr Thr Gly Gly Pro Ile Thr Tyr Ser Thr Tyr Gly Lys 260 265 270 TTC CTT GCC GAC GGT GGA TGC TCC GGG GGC GCC TAT GAC ATC ATA ATA 864 Phe Leu Ala Asp Gly Gly Cys Ser Gly Gly Ala Tyr Asp Ile Ile Ile 275 280 285 TGT GAC GAA TGC CAC TCA ACT GAC TGG ACA ACC ATC TTG GGC ATC GGC 912 Cys Asp Glu Cys His Ser Thr Asp Trp Thr Thr IleLeu Gly Ile Gly 290 295 300 ACA GTC CTG GAT CAG GCA GAG ACG GCT GGA GCG CGG CTC GTC GTG CTC 960 Thr Val Leu Asp Gln Ala Glu Thr Ala Gly Ala Arg Leu Val Val Leu 305 310 315 320 GCC ACC GCC ACG CCT CCG GGA TCG ATC ACC GTG CCA CAC CCC AAC ATC 1008 Ala Thr Ala Thr Pro Pro Gly Ser Ile Thr Val Pro His Pro Asn Ile 325 330 335 GAG GAA GTG GCC CTG TCC AAC ACT GGG GAG ATT CCC TTC TAT GGC AAA 1056 Glu Glu Val Ala Leu Ser Asn Thr Gly Glu Ile Pro Phe Tyr Gly Lys 340 345 350 GCC ATC CCC ATT GAG GCC ATC AAG GGG GGA AGG CAT CTC ATC TTC TGC 1104 Ala Ile Pro Ile Glu Ala Ile Lys Gly Gly Arg His Leu Ile Phe Cys 355 360 365 CAT TCC AAG AAG AAG TGT GAC GAG CTC GCC GCA AAG CTG ACA GGC CTC 1152 His Ser Lys Lys Lys Cys Asp Glu Leu Ala Ala Lys Leu Thr Gly Leu 370 375 380 380 GGA CTC AAT GCT GTA GCG TAT TAC CGG GGT CTC GAT GTG TCC GTC ATA 1200 Gly Leu Asn Ala Val Ala Tyr Tyr Arg Gly Leu Asp Val Ser Val Ile 385 390 395 400 CCG ACT AGC GGA GAC GTC GTT GTC GTG GCA ACA GAC GCT CTA ATG ACG 1248 Pro Thr Ser Gly Asp Val Va l Val Val Ala Thr Asp Ala Leu Met Thr 405 410 415 GGC TTT ACC GGC GAC TTT GAC TCA GTG ATC GAC TGC AAC ACA TGT GTC 1296 Gly Phe Thr Gly Asp Phe Asp Ser Val Ile Asp Cys Asn Thr Cys Val 420 425 430 ACC CAG ACA GTC GAT TTC AGC TTG GAT CCC ACC TTC ACC ATT GAG ACG 1344 Thr Gln Thr Val Asp Phe Ser Leu Asp Pro Thr Phe Thr Ile Glu Thr 435 440 445 ACA ACC GTG CCC CAA GAC GCG GTG TCG CGC TCG CAG CGG CGA GGT AGG 1392 Thr Thr Val Pro Gln Asp Ala Val Ser Arg Ser Gln Arg Arg Gly Arg 450 455 460 ACT GGC AGG GGC AGG AGT GGC ATC TAC AGG TTT GTG ACT CCA GGA GAA 1440 Thr Gly Arg Gly Arg Ser Gly Ile Tyr Arg Phe Val Thr Pro Gly Glu 465 470 475 480 480 CGG CCC TCA GGC ATG TTC GAC TCC TCG GTC CTG TGT GAG TGC TAT GAC 1488 Arg Pro Ser Gly Met Phe Asp Ser Ser Val Leu Cys Glu Cys Tyr Asp 485 490 495 GCA GGC TGC GCT TGG TAT GAG CTC ACG CCC GCT GAG ACT ACA GTC AGG 1536 Ala Gly Cys Ala Trp Tyr Glu Leu Thr Pro Ala Glu Thr Thr Val Valg 500 505 510 TTG CGG GCT TAC CTG AAT ACA CCA GGG TTG CCC GTC TGC CAG GAC CAT 1584 Leu Arg Ala Tyr Leu Asn Thr Pro Gly Leu Pro Val Cys Gln Asp His 515 520 525 CTG GAG TTC TGG GAA AGC GTC TTC ACA GGC CTC ACC CAC ATA GAT GCC 1632 Leu Glu Phe Trp Glu Ser Val Phe Thr Gly Leu Thr His Ile Asp Ala 530 535 540 CAC TTC CTG TCC CAA ACC AAG CAG GCA GGA GAC AAC TTC CCC TAC CTG 1680 His Phe Leu Ser Gln Thr Lys Gln Ala Gly Asp Asn Phe Pro Tyr Leu 545 550 555 560 560 GTG GCA TAC CAA GCC ACG GTG TGC GCC AGG GCT CAG GCT CCA CCT CCA 1728 Val Ala Tyr Gln Ala Thr Val Cys Ala Arg Ala Gln Ala Pro Pro Pro 565 570 575 TCG TGG GAT CAA ATG TGG AAG TGT CTC ATA CGG CTT AAA CCT ACG CTG 1776 Ser Trp Asp Gln Met Trp Lys Cys Leu Ile Arg Leu Lys Pro Thr Leu 580 585 590 CAC GGG CCA ACA CCC CTG CTG TAT AGG CTA GGA GCC GTT CAA AAT GAG 1824 His Gly Pro Thr Pro Leu Leu Tyr Arg Leu Gly Ala Val Gln Asn Glu 595 600 605 ATC ACC CTC ACA CAT CCC ATA ACC AAA TTC GTC ATG GCA TGC ATG TCG 1872 Ile Thr Leu Thr His Pro Ile Thr Lys Phe Val Met Ala Cys Met Ser 610 615 620 GCC GAC CTG GAG GTC GTC ACT 1893 Ala Asp Leu Glu Val Val Thr 625 630

SEQ ID NO: 2 Sequence length: 4 Sequence type: amino acid Topology: linear Sequence type: peptide sequence Arg Arg Arg Gly 1

SEQ ID NO: 3 Sequence length: 5 Sequence type: amino acid Topology: linear Sequence type: peptide Sequence Asp Gln Asp Leu Val 15

SEQ ID NO: 4 Sequence length: 8 Sequence type: amino acid Topology: linear Sequence type: peptide sequence Asp Gln Asp Leu Val Gly Trp Pro 15

SEQ ID NO: 5 Sequence length: 18 Sequence type: nucleic acid Number of strands: single strand Topology: linear Sequence type: other nucleic acid (synthetic DNA) Sequence ATTCACCTGA CTGACGAC 18

SEQ ID NO: 6 Sequence length: 18 Sequence type: nucleic acid Number of strands: single-stranded Topology: linear Sequence type: other nucleic acid (synthetic DNA) Sequence CCCTGATATT CGTCAGCG 18

SEQ ID NO: 7 Sequence length: 12 Sequence type: amino acid Topology: linear Sequence type: peptide sequence Gln Asp Leu Val Gly Trp Pro Ala Pro Pro Gly Ala 1510

SEQ ID NO: 8 Sequence length: 12 Sequence type: amino acid Topology: linear Sequence type: peptide sequence Leu Gly Tyr Ala Trp Ala Arg His Gly Ala Ala Thr 1510

SEQ ID NO: 9 Sequence length: 12 Sequence type: amino acid Topology: linear Sequence type: peptide sequence Glu Ser Arg Gly Ser Glu Arg Gly Trp Arg Thr Gln 1510

SEQ ID NO: 10 Sequence length: 16 Sequence type: Amino acid Topology: Linear Sequence type: Peptide sequence Ala His Cys Gly Trp Glu Tyr Leu Gly Trp Pro Ser Gly Leu Cys Leu 1 5 10 15

SEQ ID NO: 11 Sequence length: 16 Sequence type: Amino acid Topology: Linear Sequence type: Peptide sequence Trp Cys Gly Pro Gly Trp Pro Leu Ile Leu Lys Asp Asp Met Lys Ala 1 5 10 15

SEQ ID NO: 12 Sequence length: 12 Sequence type: amino acid Topology: linear Sequence type: peptide sequence Pro Asp Arg Arg Pro Gly Trp Arg Thr Pro Pro Arg 1 5 10

SEQ ID NO: 13 Sequence length: 12 Sequence type: amino acid Topology: linear Sequence type: peptide sequence Val Pro Gly Trp Pro Gln Asp His Ser Arg Asp Asp 1 5 10

SEQ ID NO: 14 Sequence length: 15 Sequence type: amino acid Topology: linear Sequence type: peptide sequence Trp Cys Gly Pro Ala Trp Pro Ser Val Gly Ala Ser Cys Ser Ala Ser 1 5 10 15

SEQ ID NO: 15 Sequence length: 16 Sequence type: Amino acid Topology: Linear Sequence type: Peptide sequence Trp Cys Gly Pro Gly Trp Pro Tyr Trp His Tyr Pro Pro Met Thr Asp 1 5 10 15

SEQ ID NO: 16 Sequence length: 16 Sequence type: Amino acid Topology: Linear Sequence type: Peptide sequence Trp Cys Gly Pro Asn Gly Trp Pro Ser
Asn Gln Trp Ile Gln Tyr His 15
10 15

SEQ ID NO: 17 Sequence length: 16 Sequence type: Amino acid Topology: Linear Sequence type: Peptide sequence Trp Cys Gly Pro Gly Trp Arg Thr Gly Ala Ala Arg His Leu Gly Ser 1 5 10 15

SEQ ID NO: 18 Sequence length: 16 Sequence type: Amino acid Topology: Linear Sequence type: Peptide sequence Ile Arg Arg Thr Val Gly Arg Gly Trp Pro Val Asp Gly Leu Cys Leu 1 5 10 15

SEQ ID NO: 19 Sequence length: 16 Sequence type: Amino acid Topology: Linear Sequence type: Peptide sequence Trp Cys Gly Pro Gly Trp Ala Ser Gly Ser Thr Gln Met Leu Asp Ala 1 5 10 15

SEQ ID NO: 20 Sequence length: 16 Sequence type: amino acid Topology: linear Sequence type: peptide sequence Trp Cys Gly Pro Gly Trp Pro Gln Val Gly Gln Thr Gly Ile Pro Phe 1 5 10 15

SEQ ID NO: 21 Sequence length: 16 Sequence type: Amino acid Topology: Linear Sequence type: Peptide sequence Gly Met Ala Ser Leu Thr Pro Gly Trp Arg Ile Ala Gly Leu Cys Leu 1 5 10 15

SEQ ID NO: 22 Sequence length: 12 Sequence type: amino acid Topology: linear Sequence type: peptide sequence Thr Glu Ser Ala Ala Ser Gly Trp Pro Gly Val Thr 1510

SEQ ID NO: 23 Sequence length: 15 Sequence type: Amino acid Topology: Linear Sequence type: Peptide sequence Ala Asp Val Ile Pro Val Arg Arg Arg Gly Asp Ser Arg Gly Ser 1 5 10 15

SEQ ID NO: 24 Sequence length: 12 Sequence type: amino acid Topology: linear Sequence type: peptide sequence Ser Gln Gly Gly Arg Thr Ser Pro Arg Arg Arg Glu 1510

SEQ ID NO: 25 Sequence length: 12 Sequence type: amino acid Topology: linear Sequence type: peptide sequence Thr Lys Met Gly Ile Gly Thr Arg Arg Gly Trp Ile 1 5 10

SEQ ID NO: 26 Sequence length: 12 Sequence type: Amino acid Topology: Linear Sequence type: Peptide sequence Gln Gly Arg Arg Gly Leu Val Thr Phe Trp Leu Arg 1510

SEQ ID NO: 27 Sequence length: 12 Sequence type: amino acid Topology: Linear Sequence type: Peptide sequence Glu Val Ala Arg Arg Gly Gly Phe Trp Leu Leu Arg 1 5 10

SEQ ID NO: 28 Sequence Length: 12 Sequence Type: Amino Acid Topology: Linear Sequence Type: Peptide Sequence Pro Arg Arg Arg Leu Gly Thr Leu Glu Tyr Ser Ala 150

SEQ ID NO: 29 Sequence length: 12 Sequence type: amino acid Topology: linear Sequence type: peptide sequence Val Arg Leu Pro Phe Val Met Trp Arg Arg Ala Ile 1 5 10

SEQ ID NO: 30 Sequence length: 12 Sequence type: Amino acid Topology: Linear Sequence type: Peptide sequence Lys Gly Gly Ser Pro Arg Glu Arg Met Gly Leu Thr 1510

SEQ ID NO: 31 Sequence length: 12 Sequence type: amino acid Topology: Linear Sequence type: Peptide sequence Leu Glu Asp Ala Arg Glu Arg Leu Glu Ser Ser Gly 1 5 10

SEQ ID NO: 32 Sequence length: 18 Sequence type: amino acid Topology: linear Sequence type: peptide sequence Gln Met Tyr Thr Asn Val Asp Gln Asp
Leu Val Gly Trp Pro Ala Pro 15
10 15 Pro Gly

SEQ ID NO: 33 Sequence length: 12 Sequence type: amino acid Topology: Linear Sequence type: Peptide sequence Gly Asn Leu Lys Ala Asp Gln Asp Leu Val Ala Gly 1 5 10

SEQ ID NO: 34 Sequence length: 12 Sequence type: amino acid Topology: Linear Sequence type: Peptide sequence Ala Glu Met Gln Lys Ala Asp Arg Asp Leu Val Val 150

SEQ ID NO: 35 Sequence length: 12 Sequence type: Amino acid Topology: Linear Sequence type: Peptide sequence Gln Asp Ala Ser Asp Leu Asp Leu Val Gln Arg Leu 1510

SEQ ID NO: 36 Sequence length: 12 Sequence type: amino acid Topology: Linear Sequence type: Peptide sequence Gly Ile Gly Asp Ala Asp Leu Val Arg Pro Val Val 1510

SEQ ID NO: 37 Sequence length: 12 Sequence type: amino acid Topology: Linear Sequence type: Peptide sequence Asp Ser Asp Leu Ile Lys Glu Gly Gly Ala Glu Thr 1510

SEQ ID NO: 38 Sequence length: 12 Sequence type: amino acid Topology: Linear Sequence type: Peptide sequence Glu Ala Trp Gly Gly Asp Leu Asp Leu Ile Gly Ser 1510

SEQ ID NO: 39 Sequence length: 12 Sequence type: amino acid Topology: Linear Sequence type: Peptide sequence Leu Thr Ile Ala Gly Pro Asp Val Asp Leu Met Ala 1510

SEQ ID NO: 40 Sequence length: 12 Sequence type: amino acid Topology: Linear Sequence type: Peptide sequence Asn Asn Val Asp Ser Asp Leu Leu Val Pro His Gly 1 5 10

SEQ ID NO: 41 Sequence length: 12 Sequence type: amino acid Topology: Linear Sequence type: Peptide sequence Met Val Met Cys Glu Ser Gly Val Asp Leu Val Pro 1 5 10

SEQ ID NO: 42 Sequence length: 12 Sequence type: amino acid Topology: Linear Sequence type: Peptide sequence Gln Asn Asp Leu Ile Glu Pro Gly Glu Pro Ser Cys 1510

SEQ ID NO: 43 Sequence length: 12 Sequence type: amino acid Topology: Linear Sequence type: Peptide sequence Asp Tyr Glu Gly Ser Ser Ala Ala Asp Leu Gly Leu 1510

SEQ ID NO: 44 Sequence length: 24 Sequence type: Nucleic acid Number of strands: Single strand Topology: Linear Sequence type: Other nucleic acid (synthetic DNA) Sequence ATGTACTCAA AGCGGACGGG GCGA 24

SEQ ID NO: 45 Sequence length: 21 Sequence type: nucleic acid Number of strands: single-stranded Topology: linear Sequence type: other nucleic acid (synthetic DNA) Sequence TTGCCCTGAT ATTCGTCAGC G 21

SEQ ID NO: 46 Sequence length: 5 Sequence type: amino acid Topology: Linear Sequence type: Peptide sequence Asp Xaa Asp Leu Val 15

SEQ ID NO: 47 Sequence length: 8 Sequence type: amino acid Topology: Linear Sequence type: Peptide sequence Asp Xaa Asp Leu Val Gly Trp Pro 15

[Brief description of the drawings]

FIG. 1 is a diagram showing the hepatitis C virus gene structure and various proteins encoded thereby.

FIG. 2 is a diagram showing a conventional antigenic site used in an HCV antibody test method.

FIG. 3 is a view showing a portion where a DNA encoding a random peptide in a random peptide library is present, a DNA base sequence around the portion, and arrangement of primers.

FIG. 4 is a diagram showing a nucleotide sequence of a sequencing primer in a random peptide library.

FIG. 5 is a diagram comparing the amino acid sequences of peptides capable of binding to the antibody of mAb 7E3.

FIG. 6 is a diagram comparing the amino acid sequences of peptides that can bind to the antibody of mAb 7E9.

FIG. 7 is a diagram comparing the amino acid sequences of peptides capable of binding to the antibody of mAb 8D4.

FIG. 8: Selected clones # 35, 38, 53, 121
An electrophoresis photograph comparing the results of Western blotting against mAb 8D4 antibody of the entire library before and after selection.

FIG. 9 is a diagram schematically showing epitope positions of mAb 7E9 antibody, mAb 8D4 antibody, and mAb 7E3 antibody.

FIG. 10. In the HCV strain BK strain, mAb 7E9 antibody, mAb
FIG. 9 is a view showing positions corresponding to epitope positions of the b8D4 antibody and the mAb 7E3 antibody.

FIG. 11 is a diagram illustrating an overview of a random peptide library.

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

[Procedure amendment]

[Submission date] October 27, 1997

[Procedure amendment 2]

[Document name to be amended] Drawing

[Correction target item name] Fig. 8

[Correction method] Change

[Correction contents]

FIG. 8

──────────────────────────────────────────────────続 き Continued on the front page (51) Int.Cl. ⁶ Identification code FI C12P 21/08 C12N 15/00 C

Claims (3)

[Claims] 1. The following amino acid sequence (I): Gly Trp
Pro, amino acid sequence (II): Arg Arg Arg Gly and amino acid sequence (III): Asp Xaa Asp Leu Val (Xaa
Represents any natural amino acid), the amino acid residue 12 comprising at least one amino acid sequence selected from the group consisting of
An antigenic peptide derived from the hepatitis C virus comprising the above chain peptide. 2. The following amino acid sequence (IV): Asp Xaa
The antigen peptide according to claim 1, comprising a chain peptide having 12 or more amino acid residues containing Asp Leu Val Gly Trp Pro (Xaa represents any natural amino acid). 3. An antibody test agent comprising the antigen peptide according to claim 1 or 2.