JPH07504562A - Hepatitis C virus peptide - Google Patents

Abstract

(57) [Summary] This bulletin contains application data before electronic filing, so abstract data is not recorded.

Description

[Detailed description of the invention]

Hepatitis C Virus Peptides The present invention provides novel peptides capable of binding to antibodies specific for parenterally transmitted non-A, non-B hepatitis (PT-NANBH), in particular (HCV), and the use of such peptides. for use in HCV detection immunoassays or vaccines for its prevention. related. Non-A, non-B hepatitis (NANBH) is by definition an exclusionary diagnosis and has generally been used to describe cases of viral hepatitis infections in humans that are not caused by hepatitis A or hepatitis B. . PT-NANBH virus also. It has also been called hepatitis C virus (HCV). Although the infectious cause of most of these cases has not been confirmed, based on clinical and epidemiological grounds, as reviewed by 5hin et al. (Prog, Liver Dis. 19B6.8.433-452), , many factors are responsible for this It has been thought that In the United States alone, up to 10% of blood transfusions result in NANBH. This has become a serious problem. CB-A-2239245 discloses the nucleotide and polypeptide sequences of the virus body that causes post-transfusion NANBH (PT-NANBH), and that the core region of the virus body that causes PT-NANBH has its nucleotide and peptide sequences. What is within is revealed. Disclosures that cause PT-NANBH The core region of the virus body was not analyzed in CB-A-2239245. A substantial part of the core region was used in one recombinant polypeptide, namely BHC-11, as disclosed in CB-A-2239245. The virus that causes PT-NANBH disclosed in GB-A-2239245 also covers a substantial area of the core region of the virus body. A British patent application filed on February 21, 1992 under the name Welcome Foundation on Lim1ted. It was also used to detect HCV infection in Application No. 9203803.3. EP-A-0445423 contains at least one epitope of an HCV antigen. By contacting a sample with a polypeptide having Anti-HCV antigen An assay for detecting the presence of a body is disclosed. Estimated core area of HCV? These polypeptides are used. Specific epitope regions of polypeptides are identified. Not yet. The present inventors surprisingly discovered that several dots within the N-terminal region of the HCV core protein. We discovered that the main one is an epitope. Moreover, most serum molecules that react with the N-terminal region of the core protein react with only a single domain or only a single domain. was found to react with any combination of epitope domains in Ike. This single domain was named the immunodominant domain. This immunologically dominant epitope was isolated by the inventors. Accordingly, the present invention provides an HCV virus vector containing the following sequence (SEQ ID NO: 1 and 2): Tyr-Leu-Leu-Pro-Arg-Arg (YLLPRR). Serve petit de. This sequence is found at residues 35-40 of the core protein. The present invention also includes: The above page DNA sequences encoding peptides are provided. Even if the DNA sequence is synthesized, it is It may also be a loan. Preferably, the DNA sequence is the sequence listed in SEQ ID NO. It is. When compared to the natural peptides mentioned above, the assay sensitivity and Novel synthetic peptides with improved specificity and specificity were also synthesized. In other words, the present invention also has seven aspects. General formula I X, -X, -X3-X4-X6-X, (SEQ ID NO: 3) I Syn, aspartic acid, glutamic acid, leucine or isoleucine. X2 is leucine, cysteine, aspartic acid or glutamic acid. X3 has leucine and valine. X4 is proline or methionine. Xs is arginine, aspartic acid, glutamic acid, proline, cysteine. Phenylalanine, isoleucine, asparagine, glutamine, or methionine It is. X6 is arginine, aspartic acid, glutamic acid, serine, or cysteine. However, this peptide is not L Tyr Leu-Leu-Pro-Arg-Arg (SEQ ID NO: 1 and 2)]. The present invention also includes: DNA sequences encoding peptides of general formula I are provided. This arrangement The sequence is easily ascertained considering the degeneracy of the genetic code and codon usage. The peptides of the invention may be used individually, as part of a larger peptide, and/or in association with other peptides from the HCV genome, a heterologous genome, or topographically related. It can be used as a peptide mixture with other peptides. The present invention also includes: There is provided a peptide as defined above, namely a peptide consisting of SEQ ID NOs: 4 and 5 and SEQ ID NOs: 6 and 7. The present invention also includes: DNA sequences encoding the listed peptides are provided. Preferably, the DNA sequences are as listed in SEQ ID NOs: 4 and 6. The present invention also provides an expression vector comprising a DNA sequence as defined herein. provide These vectors express their DNA sequences in suitable hosts. Peptides as defined herein can be produced. Expression vectors usually contain DNA sequences that effect expression of the DNA sequence in a suitable host. Contains clause elements. These elements vary depending on the host but typically include a promoter, a ribosome binding site, translation initiation and termination sites, and a transcription termination site. Examples of such vectors include plasmids and viruses. is included. Expression vectors of the invention include both extrachromosomal vectors and vectors inserted into the chromosome of the host cell. For use in E. coli If desired, one expression vector may contain one DNA sequence of the present invention, for example, Either the 5- or 3'-end of the DNA sequence encoding lactosidase (or) can be included, for example, as a fusion linked to the 3'-end of the DNA sequence encoding the trpE gene. For use in the insect baculovirus (AcNPV) system, the DNA sequence may optionally be co-coated with polyhetrin. fused to the array to be coded. The present invention also provides for transfection by one of the expression vectors defined herein. Provide a host cell that has been incubated with a host cell. Examples of host cells for use in accordance with the invention include prokaryotic and eukaryotic cells such as bacteria, yeast, mammalian and insect cells. Particular examples of such cells include E. coli, S. cerevisiae, P. pastoris, Chinese hamster ovary and mouse ovary, and 5podoptera frugiperda and Tricoplusiani. Host cell selection Although the choice depends on many factors, post-translational modifications of HCV viral peptides are important. eukaryotic hosts are preferred. The present invention also provides a method for producing a peptide as defined herein. The DNA sequence defined herein is isolated from the HCV genome or synthesize a DNA sequence encoding a peptide as defined herein, or generate a DNA sequence encoding a peptide of formula 1 and place the DNA sequence in an expression vector capable of expressing it in a suitable host. Insert into 1 and host with expression vector. A method is provided comprising transforming a host cell, culturing the transformed host cell, and then isolating the peptide. The DNA sequence encoding this peptide can be synthesized using standard procedures. The desired DNA sequence obtained as described above is transcribed into an expression vector using known standard techniques. - can be inserted into the Expression vectors are usually cut using restriction enzymes and DNA sequences inserted using blunt-end or sticky-end ligation. Cleavage is usually performed at a conveniently located restriction site in the expression vector such that when the DNA sequence is inserted, it is under the control of functional elements of the DNA that affect its expression. Transformation of host cells can be performed by standard techniques. expression vector to identify which transformants have been validly populated and which have not. For this purpose, some kind of phenotypic marker is usually used. Cultivation of transformed host cells and isolation of the desired peptide can also be performed using standard techniques. The peptides of the invention can be prepared by synthetic methods or by recombinant DNA technology. Pepti Preferably, the code is synthesized using an automatic synthesizer. Antibodies specific for the batches of the invention can be produced using peptides. The antibody may be polyclonal or monoclonal. Antibodies can be used for quality control testing of batches of peptides, for purification of peptides or virus lysates. It can be used for pittope mapping, for the detection of antibodies as conjugates in competitive assays when labeled, and for antigen detection assays. Polyclonal antibodies against batches 1 to 1 of the invention can be prepared by injecting the peptide into a pg mammalian host, such as a mouse, rat, sheep or rabbit, optionally coupling the peptide with a carrier to enhance the immune response, and thus It is obtained by collecting the produced antibodies. Peptides are generally administered in the form of an injectable preparation in which the peptide is mixed with a physiologically acceptable diluent. Adjuvants such as Freund's complete adjuvant (FCA) or Freund's incomplete adjuvant (FIA) can also be included in the formulation. The formulation is typically injected into the host over a suitable period of time, and plasma samples are taken at appropriate intervals to assay for anti-HCV viral antibodies. Once a suitable level of activity is obtained, the host is bled. The antibodies are then extracted and purified from the plasma using standard techniques such as protein A or ion exchange chromatography. Monoclonal antibodies against the peptides of the present invention can be used in immortalized cell lines and their viruses. by fusion with cells that produce antibodies against the same or topographically related peptides and culturing the fused immortalized cell line. Typically, a non-human mammalian host, such as a mouse or rat, is inoculated with the peptide. host antibody response After sufficient time has elapsed for the antibody to increase, the antibody-producing cells, such as spleen cells, are removed. Cells of an immortalized cell line, such as a mouse or rat myeloma cell line, are fused with antibody-producing cells, and the resulting fused cells are screened to identify cell lines, such as hybridomas, that secrete the desired monoclonal antibody. Cultivate the fused cell line and proceed as in the case of polyclonal antibody purification from the culture medium. Purify monoclonal antibodies in the same manner. A diagnostic assay based on the present invention can be used to determine the presence or absence of HCV infection. Wear. Here are the first ones again. It can be used in the treatment of such infections, for example in monitoring interferon therapy. Assays for the diagnosis of viral infections can take essentially three different approaches in connection with the detection of viral nucleic acids, viral antigens or viral antibodies. Viral nucleic acid is generally considered the best indicator of the presence of the virus itself. We can expect confirmation of materials that may be infected. however. Nucleic acid detection typically involves detection of antigens or antibodies, as the levels of the target are very low. It's not easy to get out. Viral antigens serve as markers of the presence of viruses and Used as an indicator of stainability. For some viruses, the amount of antigen present in a sample is very small (and therefore difficult to detect; detection of antibodies is, in effect, difficult because the host's immune system amplifies the response to infection by producing large amounts of circulating antibodies). The nature of the antibody response is often clinically useful, e.g. Antibodies in the 1gM class are an indicator of recent infection and are also directed against specific viral antigens. The response may involve clearance of the virus. In other words, viral sensation The actual approach taken to diagnose infection will depend on the specific environment and information provided. That will happen. In the case of HCV, one diagnostic assay can be performed using any of these three approaches. It is also realized by this. Assay methods for HCV diagnosis using detection of viral nucleic acids include hybridizing viral RNA present in the virus or cDNA synthesized from such viral RNA with a DNA sequence corresponding to the nucleotide sequence of SEQ ID NO: 1.4 or 6 or a DNA sequence encoding a peptide of formula I; Screening the generated nucleic acid hybrids to identify HCV viral nucleic acids This method consists of Application of this method is typically due to the presence of high levels of viral RNA. There is a possibility that Limited to test samples of appropriate tissues, such as liver biopsy samples. A DNA sequence corresponding to the nucleotide sequence of SEQ ID NO: 1.4 or 6 or encoding a peptide of formula I can be used as an oligonucleotide or Alternatively, it may be in the form of a cDNA sequence, and may be contained within a plasmid if desired. Screening of nucleic acid hybrids is preferably performed using labeled DNA sequences. Preferably, the peptide of the invention is part of an oligonucleotide. In this case, the label is too close to the binding site of the peptide to Position it at a sufficient distance from the peptide so as not to interfere with its binding to the acid. further characterize the hybrid, thereby further screening of one type or another to identify viral nucleic acids. You can also add one or more rounds to the round. Hybridization and The screening and screening steps are performed according to procedures known in the art. The present invention also includes: i) A DNA sequence corresponding to the nucleotide sequence of SEQ ID NO: 1.4 or 6 or is a labeled oligonucleotide consisting of a DNA sequence that coats the peptide 2, and optionally. iI) Wash solution 2 reaction buffer and substrate if the label is an enzyme. A test kit for detecting HCV viral nucleic acid is provided. Also in the test kit. It is advantageous to include a positive control sample to facilitate identification of viral nucleic acids. In assay methods for HCV diagnosis using detection of viral antigens or antibodies, one test sample is prepared using a peptide of the present invention or a polyclonal clone directed against the peptide. The presence of antigen-antibody binding within the test sample is determined. For this purpose one peptide as defined herein, or a polyclonal or monoclonal antibody thereto. and whether the test sample contains antibody or antigen binding, respectively. A test kit can be provided comprising means for determining whether exam sun The pull is performed on appropriate tissue and physiological samples as described above for the detection of viral nucleic acids. Can be collected from biological fluids. If physiological fluids are used, optionally Viral antigens or antibodies can also be concentrated. A variety of assay formats can be employed. The peptide selectively targets previously captured antibodies to selectively capture antibodies against HCV from solution. can be used to identify antibodies or to capture and label antibodies. moreover. Peptides can also be used in a variety of homogeneous assay formats in which antibodies reacted with the peptide are detected in solution without phase separation. For assay types in which peptides are used to capture antibodies from solution, the peptides are placed on a solid surface. Methods of immobilization on surfaces are included. This surface must be cleanable in some way. Must be. Examples of suitable surfaces include various polymers (molded into microtiter wells, beads, various dipsticks, suction tips, poles, and optical devices) with particle sizes typically between 0.02 and 5 microns. particles (e.g. latex) cells, solubilized red blood cells, bacteria or fungal cells. Spores, gold or other metal sols or (metal-containing sols, and proteinaceous colloids), membranes (e.g., nitrocellulose, paper, cellulose acetate, and multilayer high surface area membranes of organic or inorganic materials). It will be done. Attachment of peptides to surfaces is achieved by passive adsorption from optimized solutions containing surfactants, solvents, salts and/or chaotropes, or by active chemical bonding. Active bonding involves various reactive or activatable functionalities exposed on two surfaces. Functional groups (e.g. condensing agents; active acid esters, halides and anhydrides; amino; hydroxyl or carboxyl groups; sulfhydryl groups; carbonyl groups; dia zo group or unsaturated group). Optionally, active binding The rus peptide may be chemically coupled to a protein (which itself is attached to a surface either passively or via active bonding) by any of a variety of methods, such as through albumin or casein. The use of proteins in this method confers advantages due to isoelectric point, charge, hydrophobicity or other physicochemical properties. Viral peptides may be attached to a surface (usually, but not necessarily a membrane) after electrophoretic separation of the reaction mixture, eg, immunoprecipitation. The surface with the peptide is brought into contact (reacted) with the test sample for the time required for one reaction. Excess sample is optionally removed by any of a variety of methods (e.g., washing, centrifugation, filtration, magnetic or capillary action), and the captured antibodies are detected by any means that provides a detectable signal. . For example, this may include the above-mentioned labeled molecules or particles (such as Protein A or Protein A) that react with the capture antibody. anti-immunoglobulin subtype; rheumatoid factor; or antibodies to its peptides used in a competitive or blocking manner); can be achieved by the use of any molecule containing the epitope contained in the peptide. will be accomplished. Detectable signals include optical or radioactive or physicochemical signals, which directly target molecules or particles, such as dyes, radiolabeled electroactive species, or magnetically coupled This can be provided by labeling the molecule or particle indirectly with an enzyme that is itself capable of causing some kind of measurable change. Alternatively, if one surface is in the form of particles, the sogenals can be detected, for example using aggregation or via diffraction or birefringence effects. You can get it. Assays in which the peptide itself is used to label already captured antibodies require labeling the peptide in some form that makes it detectable. The label can be a direct label by chemical or passive attachment, such as a radiolabel for the peptide, a magnetic resonance species 2 particle or an enzyme label, or it can itself be a peptide. Indirect labeling may also be achieved by attachment of any form of label to molecules that react with tide. Chemical attachment of the label to the peptide can be achieved by attaching residues already present on the peptide, such as amino acids. or directly via an intervening group such as a maleimide group. Wear. Antibody capture involves capturing bound specific antibodies or immune complexes on any of the surfaces described. By any reagent that can achieve passive or active adsorption that results in coalescence. will be carried out. In particular, the capture of antibodies can be carried out using anti-one-type or anti-immunoglobulin subclass antibodies. rheumatoid factor, protein A, G, etc., or their peptides. Any molecule containing the epitope contained in the compound can be used. The labeled peptide is such that its binding to the specific molecules on the surface exemplified above can be used in a competitive binding mode that is blocked by the antigen in the sample. Also that This means that the antigen in the sample binds specifically or non-specifically to the above-mentioned surface, and Sometimes it can also be used in a non-specific manner to bind to the remaining valencies not used to capture the labeled peptide of a specific bivalent or multivalent molecule (eg, an antibody). In homogeneous assays, the peptide and antibody are often labeled separately and the antibody is isolated. When reacting with the recombinant peptide in the syneresis solution, the two labels e.g. thus allowing non-radioactive transfer of the captured energy to other labels. The interaction allows for appropriate detection of the excited second label or the quenched first label (eg, fluorescence magnetic resonance or enzymatic measurement). Addition of either viral peptides or antibodies into the sample limits the interaction of the label pair, resulting in a different level of signal at the detector. A suitable assay format for detecting HCV antibodies is the direct sandwich enzyme immunoassay (EIA) format. Peptide is microtiter - coated onto the wells. The test sample and enzyme-coupled peptide are added simultaneously. If HCV antibodies are present in the test sample, the Both peptides coated with peptides and enzyme-coupled peptides join to the person. Usually the same peptide is used on both sides of the sandwich. After washing, bound enzyme is detected with a specific substrate accompanied by a color change. What are the test kits for use in this type of EIA? (1) Enzyme-labeled 1 peptide as defined herein (2) Enzyme group (3) a means for providing a surface for immobilizing the peptide, and (4) optionally a wash solution and/or a buffer solution. Also, anti-human antibodies are coated onto microtiter wells and test samples are added to the wells. IgG/IgM antibody capture ELISA can also be used. It is Noh. In this case, the IgG or IgM antibodies present in the test sample are anti-human. binds to the target antibody. When a pre-labeled peptide of the invention is added to a well, the peptide Tide binds to IgG or IgM antibodies produced by HCV infection. IgG or IgM antibodies can be visualized by a label on the peptide. Thus, one peptide of the invention can be used in a number of formats, i.e. as a free peptide, in classical ELISA, competitive ELISA, membrane-bound ETA and immunochemical assays. Any assay including epidemiology can be used to detect HCV infection. The peptides of the present invention induce immunity against human HCV and can therefore be included in vaccine formulations. For this purpose, the peptide can be provided in admixture with a medically acceptable carrier. It can also be provided as a portion. Alternatively, the peptide may be attached to a specific structure, such as a ribosome or ISC0M5, if desired. Medically acceptable carriers include liquid media suitable for use as a vehicle for introducing the peptide into a patient. An example of such a liquid medium is a saline solution. The peptide may be dissolved in the carrier or suspended as a solid. stomach. Also for vaccine preparations. Adjuvants can also be included to stimulate the immune response and thereby enhance the effectiveness of the vaccine. Examples of adjuvants include hydroxide There are aluminum and aluminum phosphate. The vaccine formulation may contain a final concentration of peptide in the range of 0.01-5 mg/ml, preferably 0.03-2 mg/ml. The vaccine formulation can be placed in a sterile container, sealed and stored at low temperatures, such as 4°C, or lyophilized. To induce immunity against HCV in humans, a vaccine preparation is administered one or more times. The dose each time is O11-2 ml, preferably 0.2-1 ml. A method of inducing immunity against HCV in humans involves the use of vaccines as defined above. consisting of administering an effective amount of the Chin preparation. The present invention also provides the use of a peptide as defined herein in the manufacture of a vaccine used to induce immunity in humans against HCV. The vaccines of the invention can be administered orally and parenterally (e.g. intravenously, subcutaneously or intramuscularly). Intramuscular) injections. It can be administered by any method commonly used for administering vaccines. Treatment may include a single dose of the vaccine or multiple doses over a period of time. There are things you can do. The sequences of SEQ ID NOs: 1 to 19 cited in this description and claims are listed in the sequence listing. Ru. Next, one example of a deaf person implementing the present invention will be described. Example 1: Identification of epitope domain of HCV core protein The 97 amino acid residues of HCV core protein N-end (SEQ ID NO: 8) were mapped by PEPSCAN analysis. This method to identify epitope domains. Includes serological analysis of sequentially overlapping peptides spanning regions of interest. Ninety overlapping octamer peptides were deposited on a polypropylene bottle support such that each peptide overlapped the previous peptide by 7 N-terminal residues and the subsequent peptide by 7 C-terminal residues. was synthesized according to the manufacturer's instructions (Cambridge Research Biochemicals). Being seropositive for HCV Peptides were screened using serum or IgG purified from serum of individuals who were confirmed to have the disease. Example 2: Bebutide screening. Assay procedure 1 below was performed essentially as described in the manufacturer's instructions. Incubation times and temperatures for test antibodies varied from the indicated values. The time required for each assay was reduced. Serum: A total of 16 serum samples were assayed by PEPSCAN. These include 12 samples of serum purchased from U.S. blood donors who tested positive for antibodies to HCV during routine screening at the donation site. The remaining four sera were from patients diagnosed with non-A, non-B hepatitis and were subsequently tested to contain antibodies to HCV. This is what has been revealed. Serum can be obtained as a crude serum fraction or as caprylic acid protein. Screened as leverage. Caprylic acid purification of IgG from serum was carried out as described by Tienbuch & Audran (1969) Arch, Biochem, Biophys, 134, 279-284. So I went. Assay: The assay format consists of a blocker, test antibody, conjugate, and substrate solution. Sequential incubation of various pins in microtiter plates containing liquid Includes Immune complexes are assembled on pins from test serum with enzyme-labeled conjugates immobilized on bound anti-peptide antibodies. The pins are then dipped into the substrate. The enzyme then produces a color change, which is used to quantify the amount of bound anti-peptide antibody. Blocking: Add 200 μl of super cocktail (0.15M phosphate buffered saline pH 7.2 + 1% ovalbumin, 1% bovine serum albumin in 0.I% Tween 20) to the bottle. (min) and left at room temperature for 1 hour. Test antibodies, test antibodies are diluted between 1/200 (IgG) and l/1000 (serum) in super cocktail. The pins were then placed in this solution (175 μl/well) for 1 hour at 37°C. After this infusion, the pins were washed four times in 0.15M phosphate buffered saline pH 7.2+0, 05% Tween 20 for 10 minutes each time at room temperature. Conjugate, rabbit anti-human IgG linked to horseradish peroxidase - Dilute 1/+000 in cocktail. followed by the peptide-loaded bottle. Leave in this solution for 1 hour at room temperature, then wash the bottles again four times as described above. Substrates and substrate solutions are freshly prepared for each assay. 50 mg of 2.2'-azinobi in 100 ml of 0.1 M phosphate, 0.08 M citrate buffer pH 4.0. Add 3096 hydrogen peroxide (3-ethylhenzthiazine sulfonic acid) (ABTS) and 0.3 μl/ml 3096 hydrogen peroxide. Place the pins into wells containing substrate solution (150 μl/well) and incubate at room temperature in the dark. When sufficient color develops, remove the bottle and stop the reaction. Immediately place the plate in a spectrophotometric plate library. It is read at a wavelength of 405 nm using a radar. Next, plot the absorption value against the pin number. lot. The epitope domain is marked with an amino acid common to all positive pins in the domain. Acid continuous sequence, i.e. Positive Pin 1 Gly-Val-Tyr-Leu-Leu-Pro-Arg-Arg (SEQ ID NO. No. 9) (G V Y L L P RR) Positive pin 2 Val-Tyr-Leu-Leu-Pro-Arg-Arg-Gly (SEQ ID NO. No. 10) (V Y L L P RRG) Positive pin 3 Tyr-Leu-Leu-Pro-Arg-Arg-Gly-Pro (SEQ ID NO. No. 11) (Y L L P RRG P). In this example the epitope has the sequence Tyr Leu-Leu-Pro-Arg-Arg (YLLPRR) (SEQ ID NO: 1 and 2). After cleaning and use, bottles were diluted with cleavage buffer (1% sodium dodecyl sulfate, 0.1% 2-mercaptoester in 0.1 M sodium dihydrogen phosphate, 0.1% 2-mercaptoester) heated to 60°C. (Tanol) Cleaned by sonication. Sonicate the bottle for 30 minutes and and washed twice in distilled water at 60°C and once in boiling methanol. bottle It can then be stored sealed in a bag containing silica gel or immediately reused for the next assay. Several domains within the N-terminal region of the HCV core protein are thus epitopically linked. was identified as a group. One of these regions, an immunologically dominant epitope, was detected to a greater or lesser extent in all sera tested. Immunologically dominant epitopes are It is located at residues 35-40 of the protein and has the amino acid sequence TyrLeu-Leu-Pro-Arg-Arg (YLLPRR), SEQ ID NOs: 1 and 2. The assay results for the samples are shown in Table 1. Table 1: Absorption units for various serum Peptide SEQ ID NO: 65221 65241 65276 65234 LG GGQIVGC; 12 137 121 127 134 121GGQIV GGV 13 323 122 119 179 119 GQIVGGVY 1 4 216 112 128 165 138QIVGGVYL 15 329 125 165 152 1611VGGVYLL 16 268 126 183 186 158VGGVYLLP 17 83 144 182 23 5 169LLPRRGPR2214212998101133LPRRGPR L 23 97 94 139 145 148PRRGPRLG 24 85 73 129 109 110RRGPRLGV 25 145 85 15 0 146 123RGPRLGVR261689416799132GPRL GVRA 27 111 60 146 75 101PRLGVRAT 28 166 79 155 88 115 Amino acid residues are expressed as one-letter code (Eur, J, Biochem, 138, 9-37.1984) below t6° Lined symbols indicate immunologically dominant regions. Example 3: Production of novel mimetics of original epitopes Using the substitution net method, each constituent amino acid residue in an epitope is sequentially replaced with each other genetically fed amino acid. I changed it. For substitution net analysis, the parent sequence was subjected to epitope mapping software provided by Cambridge Research Biochemicals to generate a set of substituted peptides. This process is the 6th stage of HCV. The amino acid residue immunologically dominant core epitope was performed. Peptides were synthesized on polypropylene bottles as per the manufacturer's instructions (CRB). assay former The set was the same as in the case of PEPSCAN above. We investigated the substitution net of the core immunologically dominant epitope sequence, Tyr-Leu-Leu-Pro-Arg-Arg (YLLPRR) (SEQ ID NOs: 1 and 2), in the purchased donated human serum described above. For each position within the epitope, indicate the frequency of each substituted residue as an acceptable substitution relative to the parent sequence. Substituted residues were considered acceptable in the above cases. Based on these results, it is possible to know which substituted amino residue at each position is most likely to be an enhancing substitution. These are shown in Table 2. Underlined residues are the parent sequence. Table 2 = Parent Residue Amino Acid Relative frequency of signal torsobutton (n = 12) Highest 2nd to 5th 6th to 10th 10th or less Total-Tyrosine D 18 1 010 E281 011 L 71 1 0 9 Y 2 3 5 1 11 2-Loin: /C281011 D640 010 E164011 V650 011 4-Blower: /M45 0 0 9 P 3 River 1 05 5-Arginine CO82010 D 4 5 1 1 11 E 2 4 3 1 10 F 0 2 7 3 12 M 0 2 2 6 t. N0O6511 P154111 RO21710 6-Arginine C040610 D323 210 E 1 6 2 1 10 R215311 Example 4: Preparation of AC22 The fully protected peptide-resin was purchased from Applied Biosystems User Bulletin, No. 33, November 1990, by a nine-step solid-phase synthesis. (cal) was assembled on p-hydroxymethylphenoxymethyl (HMP) resin. boxyl 1 (proceeding from the terminal residue to the amino-terminal residue), the HMP resin was transferred to the reaction volume of an Applied Biosystems Peptide Synthesizer Model 431A. Transferred to a container. Carboxyl-terminal amino acid residues were coupled using the standard AppHed Bjosyste+++s Fastmoc cycle protocol. I set it. Unreacted coupling sites on the resin were then blocked by capping with benzoic anhydride. All subsequent amino acids were then processed using Fastmoc Cycle Pro. tocol in a stepwise manner from the carboxyl end to the amine end. I set it. The N-α-amino group of all peptides is 9-fluorophenylmethoxycarbonate. Rubonyl (Fmoc) was linked and protected. The side chain functional groups of related amino acids were protected with the following groups. Cys-Trt () lytyl) Gly-Trt () lytyl) Tyr-tBu (tertiary butyl) Arg-P+nc (2,2,5,7,8-pentamethylchroman-6-sulfo Transfer the fully protected peptide-resin to a pear-shaped flask and add ethanedithio (0.25ml), thioanisole (0.5ml), water (0.5ml), etc. and TFA (8.75ml). The mixture was stirred for 90 minutes at room temperature and then filtered through glass wool in a Pasteur pipette. Collect the filtrate The peptide precipitated when added dropwise into water-cooled ether in a centrifuge tube. centrifugation Centrifuge the tube. Aspirating the liquid left a pellet of peptide in the centrifuge tube. ether the peptide (Use a spatula to prevent pellet clumping) and collect the peptides by centrifugation. This was repeated a total of 6 times. The peptide was finally dried by centrifugation in vacuo. Example 5. Vaccine formulations Vaccine formulations can be manufactured by conventional techniques using the following components as indicated: Ru. HCV viral peptide >0.36 (alone, as a long polypeptide or or with a cocktail of other peptides) Thiomersal 0.04-0. 2mg Sodium chloride <8.5 Water <1ml

[Sequence list]

Array number = 1 Sequence length, 18 base pairs Sequence type: Nucleic acid Number of strands – single strand l-bology: linear Sequence type: cDNA Characteristics of arrays Name/symbol representing characteristics: CDS Existence position +1. ．． 18 array TACTTG’ TTG CCG CGCAGGTyr Leu Leu Pr o Arg ArgI5 Sequence number 2 Sequence length 26 amino acids sequence type diamino acid Topology: linear Sequence type: protein array Tyr　Leu　Leu　Pro　Arg　ArgI5 Sequence number 3 Sequence length 26 amino acids sequence type diamino acid topology, unknown Sequence type: Peptide Characteristics of arrays Name/symbol representing characteristics: modified-site Location: One of (1) Other information: Note = rXaa is Tyr, Asp, Glu, Leu or Il Characteristics of the array “representing e” Name/symbol representing the feature + modified-site location = one of (2) Other information: Note = rXu represents LeuI Cys, Asp or Glu. Column characteristics Name symbol representing the characteristic: modif 1ed-site Existence position = 1 of (3) One Other information: Note = "rXaa represents Leu or Val" Sequence characteristics Name/symbol representing the feature: modified-site location = one of (4) Other information: Note = rXu stands for Pro or Met” Sequence characteristics Name/symbol representing the feature: modified-site location = one of (5) Other information: Note = rXaa is Arg, Asp, Glu, Pro, Cys , Phe, Ile, Asn. "Gln or Mal" Characteristics of arrays Name/symbol representing characteristics: modif 1ed-site location = (6) one Other information: Note = rXaa is Arg, Asp, Glu, Ser or Cy "array representing s" Xaa Xaa Xaa Xu Xu Xu Sequence number, 4 Sequence length - 51 base pairs Sequence type: Nucleic acid chain number knee heavy chain Topology: linear Sequence type: cDNA Characteristics of arrays Name/symbol representing characteristics CDS Existence position: 1. ．． 51 Other information: Note = "Nucleotides 1-3 are of non-HCV origin" sequence TGCCAG ATCGTT GGT GGA GTT TACTTG ICy s Gin lie Mal Gly Gly Val Tyr LeuI 5 TTG CCG CGCAGG GGCCCT AGA TTC5Leu Pr o Arg Arg GIF Pro Arg LeuI015 Sequence number-5 Sequence length: 17 amino acids sequence type, amino acid chain number knee heavy chain Topology - linear Sequence type, protein array Cys Gln Ile Mal Gly Gly Val Tyr Leu Leu Pro Arg ArgI S 10 Gly Pro Arg Leu Sequence number: 6 Sequence length = 51 base pairs Sequence type: Nucleic acid chain number knee heavy chain topology bilinear 7 Type of sequence: cDNA Characteristics of arrays Name/symbol representing characteristics: CD5 1 Location: 1. ．． 51 Other information, note = [Nucleotides 49-51 are of non-HCV origin] Sequence CAG ATCGTT GGT GGA GTT TACTTG TTG’1 IGinlie Vat Gly Gly Val Tyr Lag Leu I5 CCG CGCAGG GGCCCT AGA TTCTGC51Pro Ar g　Arg　Gly　Pro　Arg　LeuCysU　tS Sequence number 7 Sequence length = 17 amino acids sequence type diamino acid chain number knee heavy chain Topology = linear Sequence type: protein array Gln lie Vil Gly Gly Vml Tyr Leu Leu Pro Arg Arm Gly151゜ Pro Arg Leu Cys ! 5 Sequence number 28 Sequence length: 97 amino acids sequence type, amino acid topology unknown Sequence type: Peptide array Met Ser Tbr Amn Pro Lys Pro Gin Arg Lye Thr Lys ArgI S 10 Asn Tbr Asn Arg Arg Pro Gln Asp Vil Lys Phe Pro GlyI5 20 25 Gly Gay Gln eyes Val Gly Gly Vat Tyr L eu Lea Pro＾rgArg Gly Pro Arg Leu Gly Vml Arg Ala Thr Arg Lys ThrSer Glu Arg’ Sar Gin Pro Arg Gly Arg Arg Gln Proll565 80 @5 Pro Lys Ala Arm Arg Pro Glu Gly Arg Tbr Trp Aim G1n70 Fu5 Pro Gly Tyr Pro Trp Pro Leu Tyr Gly Asn　Glu　Gly　Leugo　ss　i. Gly Trp Ala Gly Trp Lau Sequence number: 9 Sequence length 28 amino acids sequence type diamino acid topology unknown Sequence type: Peptide array Gly Val Tyr Leu Leu Pro Arg ArgI5 Sequence number = 10 Sequence length: 8 amino acids Sequence type Amino acid Topology/Unknown Sequence type: Peptide array Val Tyr Leu Leu Pro Arg Arg Gly sequence number, 11 Sequence length: 18 amino acids Sequence pear, amino acids topology, unknown Sequence type: Peptide array Tyr Leu Leu Pro Arg Arg Gly Pr. Sequence number, 12 Sequence length = 8 amino acids Sequence type Amino acid topology, unknown Sequence type: Peptide array Gly Gly Gly Gln Ile Val Gly Gly sequence number・ 13 Sequence length = 8 amino acids sequence type, amino acid Tobology: unknown Sequence type: Peptide array Gly Gly Gin Ile Val Gly GlyVal Sequence number: 1 4 Sequence length 28 amino acids sequence type diamino acid Tobology: unknown Sequence type: Peptide array Gly Gin lie Val Gly Gly Val Tyr Sequence number = 15 Sequence length = 8 amino acids sequence type diamino acid topology unknown Sequence type, peptide array Ginlie Val Gly Gly Val Tyr Leu Sequence number: 16 Sequence length 28 amino acids sequence type diamino acid topology, unknown Sequence type: Peptide array 11e Val Gly Gly Vat Tyr Leu Leu Sequence number: 17 Sequence length 28 amino acids sequence type diamino acid L.Bolozi: unknown Sequence type: Peptide array Val　Gly　Gly　Val　Tyr　Leu　Leu　Pr. Sequence number: 18 Sequence length = 8 amino acids sequence type diamino acid topology unknown Sequence type: Peptide Characteristics of arrays Name/symbol representing the sequence: Region Location: 4. ．． 8 Other information: Note = “Immunological dominant region” array Gly Gly Vat Tyr Leu Leu Pro Arg Sequence number = 19 Sequence length, 8 amino acids sequence type diamino acid topology unknown Type of sequence; peptide Characteristics of arrays Name/symbol representing array 2 Region Location: 3. , 8 Other information: Note = “Immunological dominant region” array Gly Val Tyr Leu Leu Pro Arg Arg Sequence number: 20 Sequence length 28 amino acids sequence type diamino acid Topology: unknown Type of sequence; peptide Characteristics of arrays Name/symbol representing array: Region Location: 2. ,7 Other news: Note = “Immunological dominant region” array Val Tyr Leu Leu Pro Arg Arg Gly Sequence number = 21 Sequence length = 8 amino acids sequence type diamino acid topology unknown Sequence type: Peptide Characteristics of arrays array Gly Pro Arg Leu Gly Vat Arg Ala sequence number = 28 Sequence length = 8 amino acids sequence type diamino acid topology unknown Sequence type: Peptide array Pro Arg Leu Gly Vat Arg Ala Thr Sequence number: 29 Sequence length 28 amino acids Sequence type; amino acid topology unknown Sequence type, peptide array Arg Leu Gly Val Arg Ala Thr Arg. 1Il- Continuation of N+PCT/Gil193100410 front page (51) Int, C1,6 identification symbol Internal office reference number C07K 7106 8 318-4H 14/18 8318-4H C12N 15109 C12P 21102 ZNA C9282-4B21108 9161-4B C12Q 1/68 A 9453-48GOIN 33153 D 7055 -2J331576 Z 7055-2J I

Claims (1)

[Claims] 1. array: HCV viral peptide with [sequence] (SEQ ID NO: 1 and 2) 2. General formula I X1-X2-X3-X4-X5-X6 (SEQ ID NO: 3) I [wherein, X1 is tyrosine , aspartic acid, glutamic acid, leucine or isoleucine, X2 is leucine, cysteine, aspartic acid or glutamic acid; are leucine and valine, X4 is proline or methionine, and X5 is arginine, aspartic acid, Glutamic acid, proline, cysteine, phenylalanine, isoleucine, as paragine, glutamine, or methionine; X6 is arginine, aspartic acid, glutamic acid, serine, or cysteine It is. However, this peptide does not have the formula [sequence exists] (SEQ ID NO: 1 and 2). ] a peptide with the sequence 3. Viruses having the sequences listed in SEQ ID NOs: 4 and 5 or SEQ ID NOs: 6 and 7 speptidome 4. 5. DNA sequence encoding the peptide according to "Claim 1, 2 or 3". Distribution 6. DNA sequence according to "Claim 4" listed in column number 1, 4 or 6. “Claim 4 or an expression vector containing the DNA sequence described in 5. an expression vector that allows the production of peptides by expression of its DNA in 7. 8. Host cell transformed with the expression vector according to "Claim 6". In producing the peptide according to any one of "Claims 1 to 3", SEQ ID NO: 1 , 4 or 6 is isolated from the HCV genome, or the DNA sequence listed in SEQ ID NO. Synthesize a DNA sequence encoding a peptide listed in item 1, 4 or 6, or generates a DNA sequence encoding a peptide of formula I, and this DNA sequence is suitably Insert it into an expression vector that can express it in a suitable host, and insert it into an expression vector that can express it in a suitable host. Transform host cells in a vector and culture the transformed host cells. 9. Method consisting of culturing and isolating the peptide.9. Any of “Claims 1 to 3” In producing the described peptide, it is characterized in that it is produced synthetically. Method 10. Polyclonal clone for the peptide according to any one of "claims 1 to 3" monoclonal antibody 11. In the detection method of HCV viral nucleic acid, virus present in the test sample is rus RNA or cDNA synthesized from such RNA with SEQ ID NOs: 1 and 4. or a DNA sequence corresponding to 6 or a DNA sequence encoding a peptide of formula I; Hybridization was performed, and the generated nucleic acid hybrids were screened to detect HCV infections. 12. A method comprising identifying viral nucleic acids. Method for detecting HCV virus antibodies In this case, the test sample is treated with the peptide according to any one of claims 1 to 3 or contact with the polyclonal or monoclonal antibody according to claim 10, from determining whether the test sample contains antigen-antibody binding. Method 13. In the kit for detecting HCV virus antibodies, The peptide according to any one or the polyclonal according to "Claim 10" or is a monoclonal antibody, and the test sample contains an antigen-antibody bond. A kit comprising means for determining whether 14. Any of “Claims 1 to 3” A vaccine preparation comprising the peptide described above in combination with a medically acceptable carrier. 15. comprising administering an effective amount of the vaccine formulation according to "claim 14". Method for inducing immunity against HCV in humans