KR20170077600A - A composition, kit and method for concurrent detecting of Hepatitis C virus antigen and antibody thereto - Google Patents

Abstract

Using a fusion polypeptide comprising the C-terminal fragment of HCV NS3, the hydrophilic fragment of the core protein, the hydrophilic segment of NS4, and the hydrophilic segment of NS5 to specifically bind to a polypeptide comprising a hydrophilic segment of the HCV core protein The HCV antigen of the individual and the antibody thereof can be detected simply and quickly, thereby diagnosing the hepatitis C virus.

Description

Technical Field [0001] The present invention relates to a composition, kit and method for simultaneous diagnosis of hepatitis C virus antigen and antibodies thereto,

A kit, and a method for simultaneous diagnosis of hepatitis C virus antigen and antibody.

Recently, many basic medical sciences related to medicine have been developed and various infectious pathogens that can cause diseases to humans have been found. Among them, hepatitis C virus (HCV), along with hepatitis B virus (HCV), is increasingly a problem not only in Korea but also in the world.

Hepatitis C virus (HCV) infection is a disease in which liver inflammation occurs. The blood of a patient infected with hepatitis C virus can be transmitted through wounded skin, mucosa, and blood of a normal person. Most cases of hepatitis C virus infection are not symptomatic at the early stage of infection. In case of infection with adult hepatitis B virus, chronic hepatitis is over 75%. About 12% of patients with liver cirrhosis and about 15% of patients with hepatocellular carcinoma are caused by hepatitis C virus and are the main causes of chronic hepatitis with hepatitis B virus.

However, existing hepatitis C virus detection reagents require a considerable amount of time to be detected after human infection with hepatitis C virus, and thus they can be used as a contaminated state for hepatitis C virus when used for blood and blood transfusion. The supplementary method such as the diagnosis of hepatitis molecule is used as a complementary measure for this, but the huge cost is incurred due to the cost of the reagent purchase fee for the diagnosis, the labor cost of the searcher, equipment and facilities, installation and operation expenses, Prices will also increase.

Therefore, diagnosis of hepatitis C virus is required more easily and quickly.

One aspect provides polypeptides comprising HCV core protein hydrophilic fragments and antibodies thereto.

Another aspect provides a polynucleotide encoding said polypeptide.

Another aspect provides a hybridoma cell of accession number KCLRF-BP-00354 or KCLRF-BP-00355 and an antibody that specifically binds to said polypeptide produced thereby.

Another aspect provides compositions and kits for detecting an antigen of HCV comprising an antibody that specifically binds to a polypeptide comprising a hydrophilic fragment of HCV core protein.

Another aspect is an antibody and an antibody of HCV comprising an antibody that specifically binds to said polypeptide and a fusion polypeptide comprising a C-terminal fragment of HCV NS3, a hydrophilic fragment of core protein, a hydrophilic segment of NS4, and a hydrophilic segment of NS5 A kit and a composition for simultaneous detection are provided.

Another aspect is the use of an antibody specifically binding to said polypeptide and a hepatitis B and C infection using a fusion polypeptide comprising a C-terminal fragment of HCV NS3, a hydrophilic fragment of core protein, a hydrophilic segment of NS4 and a hydrophilic segment of NS5 And provides a method for simultaneous diagnosis.

One aspect provides a polypeptide comprising a hydrophilic fragment that is an amino acid sequence selected from the hepatitis C virus (HCV) core protein.

The fragment may be a contiguous amino acid sequence selected from whole protein or peptide amino acid sequences.

"Hydrophilic" may mean that the molecule or substance is polar and has a high affinity for water, for example, the Kyte-Doolittle method (Kyte, J. and Doolittle, R. 1982, J. Mol. 105-132). A molecule or material having hydrophilicity is one that is partially hydrophilic and includes only a molecule or a portion of the material that is capable of interacting with water, and the entire portion of the molecule or material need not be hydrophilic. For example, when the amino acid sequence of the peptide includes a polar amino acid including lysine, arginine, histidine, aspartic acid, serine, threonine, glutamic acid, asparagine, glutamine, cysteine, glycine and proline, the peptide may have hydrophilicity And the hydrophilic amino acid may be included in the amino acid sequence of the peptide, so as not to lose hydrophilicity. Even if a region of the peptide is composed of a hydrophobic amino acid, it can be a region having hydrophilicity due to the influence of the adjacent hydrophilic amino acid.

The genes for HCV include genes coding for core proteins, E1 and E2, and nonstructural proteins NS1, NS2, NS3, NS4A, NS4B, NS5A and NS5B. The protein NS3 comprises two structural domains, the N-terminal domain conferred by activated serine protease activity belonging to the maturation of the viral polyprotein, and the helicase activity associated with the NTPase activity which plays a role in the replication of the viral genome C-terminal domain. The core protein is the first structural protein encoded by the HCV ORF and forms the nucleocapsid of the virus. The N-terminal of the core protein is abundant in basic amino acids and may have hydrophilic properties. Protein NS4 can be divided into NS4A and NS4B. NS4A can form NS3 and NS3-4A serine proteases. Protein NS5 can be distinguished as NS5A and NS5B, and can exist in phosphorylated or hyperphosphorylated forms.

The fragment may comprise amino acids 1, 2, 3, 4, 5, 10, 15 or 20 from the N-terminus of the core protein to amino acids 150, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 170, 175 or 180 amino acids. For example, the fragment may be a hydrophilic fragment of an HCV core protein comprising the amino acid sequence of SEQ ID NO: 1.

The amino acid sequence of the polypeptide can be suitably modified to the extent that it does not substantially change the objective function or characteristic of the original protein, for example, antigenicity. The modification of the amino acid is based on the similarity of the amino acid residue substituent, for example, hydrophobicity, hydrophilicity, charge, size, etc. For this purpose, a hodrophobic index of the amino acid can be considered. Such modifications may be, for example, substitution, deletion, addition, etc., of some amino acids, and substitutions may be conservative substitutions in particular. The term "conservative substitution" refers to a substitution that does not significantly alter the biological activity of the resulting molecule, where the substituted amino acid does not cause a significant effect on the tertiary structure or local charge state of the protein. Ala / Gly, Ala / Thr, Ser / Asn (SEQ ID NO: 2), Ala / Ser, Val / Ile, Asp / Glu, Thr / Ser, Ala / Gly, Ala / Thr and the like are known in the art. , Ala / Val, Ser / Gly, Thy / Phe, Ala / Pro, Lys / Arg, Asp / Asn, Leu / Ile, Leu / Val, Ala / Glu and Asp / Gly.

Said polypeptide having at least 60%, 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% 0.0 > 100% < / RTI > sequence identity. The polypeptides may also comprise one, two, three, four, five, six, seven, eight, nine or ten or more bases of the active site or antibody binding site in the amino acid sequence of SEQ ID NO: May have different sequences. In addition, the polypeptide may be one in which some amino acids of the amino acid sequence of SEQ ID NO: 1 are substituted with other amino acids within a range that does not change the activity of the fragment.

The polypeptide may be one with a detectable label attached thereto. The detectable label may be an optical label, an electrical label, a radioactive label, an enzyme label, or a combination thereof. The optical label may be a substance that generates fluorescence or phosphorescence. The fluorescent material may be, for example, fluorescein, rhodamine, cyanine, metal porphyrin complex, Cy-5 and Cy-3. Examples of fluororesin dyes include, but are not limited to, 6-carboxyfluorescein (6-FAM), 2 ', 4', 1,4- tetrachlorofluorescein (TET) 7 ', 1,4'-hexachlorofluorescein (HEX), 2', 7'-dimethoxy-4 ', 5'-dichloro-6-carboxydodamine (JOE) Fluoro-7 ', 8'-fused phenyl-1,4-dichloro-6-carboxyfluorescein, and 2'-chloro-7'-phenyl-1,4- . The enzyme label may be one which converts the substrate to a coloring material.

Another aspect provides an antibody or antigen-binding fragment thereof that specifically binds to a polypeptide comprising a hydrophilic fragment having a contiguous amino acid sequence selected from an HCV core protein. The term "antigen binding fragment" refers to a fragment of the immunoglobulin whole structure, and refers to a portion of a polypeptide comprising a moiety capable of binding an antigen. The antibody and its antigen-binding fragment can recognize the core protein of HCV and can be used to detect HCV.

The terms "specifically binding" or " specifically recognizing "are the same as commonly known to those of ordinary skill in the art, wherein the antigen and antibody specifically interact to form an antigen- And may also mean immunological reactions.

Among the terms or elements mentioned in the description of the antibody, those already mentioned in the description of the polypeptide are as described above.

Another aspect provides hybridoma cells with accession numbers KCLRF-BP-00354 or KCLRF-BP-00355.

In one embodiment, the antibody may be one produced in a hybridoma cell having a deposit number KCLRF-BP-00354 or KCLRF-BP-00355. The hybridoma cells can be prepared using methods known in the art. Specifically, the hybridoma cells are prepared by immunizing an animal with the polypeptide as an immunogen, fusing B cell, an antibody-producing cell derived from the animal to be immunized, with a myeloma cell to prepare a hybridoma, And selecting a hybridoma that produces a monoclonal antibody that specifically binds to the antibody. The animal to be immunized can be an animal such as a goat, a sheep, a guinea pig, a rat or a rabbit as well as the mouse used in the examples.

As a method for immunizing the immunized animal, a method well known in the art can be used. For example, when a mouse is immunized, an immunogen of 1 to 300 占 퐂 at one time is emulsified with an antigen adjuvant such as an equivalent amount of physiological saline and / or Freund's adjuvant, Or by inoculating 2-6 times every 1-5 weeks in the abdominal cavity. After the immunized animal is immunized, the spleen or lymph node is harvested after 3-10 days of the final immunization, and the B cells contained in the tissue of the spleen or lymph node are treated with a myeloma Cells. As the fusion promoter, for example, a material such as polyethylene glycol (PEG) may be used. The myeloma cells include, for example, P3U1, NS-1, P3x63. Ag 8.653, Sp2 / 0-Ag14, and rat-derived cells such as AG1 and AG2 can be used. In addition, the cell fusion method known in the art includes, for example, mixing B cells and myeloma cells in a ratio of 1: 1 to 20: 1, adding PEG having a molecular weight of 1,000-6,000 to a concentration of 10-80% Followed by incubation at 30-37 ° C for 1-10 minutes. Hybridomas producing a monoclonal antibody that specifically binds to the polypeptide can be produced by, for example, culturing in a selective medium such as a HAT medium in which only hybridomas are viable, and culturing the hybridomas in a hybridoma culture supernatant Can be selected and measured by a method such as ELISA. Finally, a hybridoma that produces a monoclonal antibody that specifically binds to the polypeptide can be produced by, for example, a method of limiting the amount of the hybridoma producing monoclonal antibody that specifically binds to the polypeptide, Lt; RTI ID = 0.0 > cloning < / RTI > The monoclonal antibody may be of the IgG1, IgG2, IgG3, IgG4, IgM, IgE, IgA1, IgA5, or IgD type.

Among the terms or elements mentioned in the description of the hybridoma cell, those already mentioned in the description of the polypeptide or the antibody are as described above.

Another aspect provides a polynucleotide encoding a polypeptide comprising a hydrophilic fragment that is an amino acid sequence selected from HCV core proteins.

As used herein, the term "polynucleotide" may refer to a polymer of deoxyribonucleotides or ribonucleotides present in single-stranded or double-stranded form. RNA genomic sequences, DNA (gDNA and cDNA) and RNA sequences transcribed therefrom, and may include natural polynucleotides as well as analogues thereof in which the sugar or base moiety is modified, have.

The polynucleotide may include a nucleotide sequence encoding the amino acid sequence of the protein complex, as well as a complementary sequence to the sequence. The complementary sequence includes a perfectly complementary sequence as well as a substantially complementary sequence, which can be obtained under stringent conditions known in the art, for example, a nucleotide sequence encoding the amino acid sequence of the polypeptide ≪ RTI ID = 0.0 > nucleotides < / RTI >

In addition, the nucleotide sequence encoding the amino acid sequence of the polypeptide may be modified. Such modifications include addition, deletion or non-conservative substitution or conservative substitution of nucleotides. The polynucleotide encoding the amino acid sequence of the polypeptide may be interpreted to include a nucleotide sequence that exhibits substantial identity to the nucleotide sequence. The above-mentioned substantial identity is determined by aligning the nucleotide sequence with any other sequence as much as possible and analyzing the aligned sequence using an algorithm commonly used in the art. The sequence is 80% or more homology, 90 Or more homology or 95% or more homology. The polynucleotide may comprise the sequence of SEQ ID NO: 8.

The production of the polypeptide can be carried out using genetic engineering techniques and chemical synthesis known in the art using the polynucleotide. The gene manipulation technique may include constructing a cloning vector or an expression vector capable of replication, transforming the host cell with the vector, and culturing the host cell to express the target protein.

Accordingly, one aspect includes constructing a recombinant expression vector into which a polynucleotide sequence encoding the above-mentioned polypeptide is inserted, transforming the host cell with the recombinant expression vector, culturing the transformed host cell, and And recovering the polypeptide expressed in the host cell, wherein the polypeptide comprises a hydrophilic fragment having a contiguous amino acid sequence selected from the HCV core protein.

The term "vector" is a means for expressing a target gene in a host cell. When introduced into a host cell, the vector is independently replicated to produce a copy of the vector and the inserted foreign DNA. The term "recombinant expression vector" is a vector into which a foreign DNA fragment is inserted for the purpose of amplification of a specific protein. The foreign DNA fragment may be a polynucleotide encoding the above-mentioned polypeptide. Methods for constructing vector systems for expression or cloning purposes are known.

The vector may comprise a control sequence operably linked to a polynucleotide sequence as defined herein. The term "regulatory sequence" refers to the nucleic acid sequence necessary to effect the expression of the coding sequence, the nature of the regulatory sequence being dependent on the host organism. The term " operatively linked "may refer to a juxtaposition by a functional combination that allows the components to function in an intended manner.

In addition to the regulatory sequences, the recombinant expression vector may further include a restriction enzyme cleavage site, a marker gene such as a drug resistance gene, a secretory signal sequence or a leader sequence. The restriction enzyme cleavage site refers to a specific base sequence that is specifically recognized and cleaved by restriction enzymes. The cleavage site may be a sequence specifically recognized by restriction enzymes such as, for example, EcoRI, BamHI, HindIII, kpnI, SalI, NotI, PstI, SmaI and XhoI. The marker gene functions as a selection marker and may be a resistance gene of a drug such as ampicillin, genomicin, carbenicillin, chloramphenicol, streptomycin, kanamycin, geneticin, neomycin, and tetracycline. The secretory signal sequence or the leader sequence is a sequence which directs the synthesized protein to a cell compartment (for example, a periplasmic space) or secretes it into an extracellular medium. The secretory signal sequence or the leader sequence includes a coding sequence of the polynucleotide sequence Lt; / RTI > Such sequences may be appropriately selected by those skilled in the art depending on the DNA to be introduced, the type of host cell, the conditions of the culture medium and the like.

The host cell may be produced by transforming or transfecting the host with the recombinant expression vector described above.

The host cell may be any prokaryotic or eukaryotic cell known in the art capable of stabilizing and subsequently cloning and expressing the recombinant vector. The prokaryote refers to a bacterium that can be transformed into a DNA or RNA molecule for expression of the protein.

Transformation of a recombinant expression vector into a host cell can be accomplished by, for example, DEAE-dextran mediated transfection, electroporation, transduction, calcium phosphate transfection ), Cationic lipid-mediated transfection, scrape loading and infection, and the like.

The cultivation of the host cells may be carried out using appropriate media and culture conditions known in the art. For example, LB medium, Ham's F10 (Sigma), MEM (Minimal Essential Medium, Sigma), RPMI-1640 (Sigma), and DMEM (Dulbecco's Modified Eagle's Medium, Sigma). If desired, hormones or other growth factors, salts, buffers, nucleotides, antibiotics, trace elements and glucose may be supplemented with known concentrations of known agents. The culture conditions, such as temperature, pH and the like, can be appropriately determined by those skilled in the art depending on the host cell selected.

A polypeptide expressed by a host cell can be secreted extracellularly by a secretory signal peptide, which in this case can be obtained by recovering it from a culture medium or medium. For example, the culture supernatant can be concentrated using a commercially available protein filter to isolate the polypeptide. However, when expressed without the secretory signal sequence, the polypeptide can be obtained directly from the cell lysate since it is present in the intracellular periplasmic space. Processes for isolating polypeptides secreted into the periplasmic space are known, and generally microparticle fragments (host cells or broken fragments) can be removed by centrifugation or ultrafiltration.

Alternatively, the polypeptides obtained from such cultures may be further purified by methods known in the art. For example, depending on the polypeptide being recovered, the polypeptide may be purified by chromatography (e.g., ion exchange, affinity, hydrophobicity and size exclusion), chromatofocusing, SDS-PAGE, fractional dissolution (e.g., ammonium sulfate precipitation ), Can be used.

Among the terms or elements mentioned in the description of the polynucleotide, those already mentioned in the description of the polypeptide, antibody or hybridoma cell are as described above.

Another aspect is a composition for detecting an HCV antigen of an individual comprising an antibody or antigen-binding fragment thereof that specifically binds to a polypeptide comprising a hydrophilic fragment having a contiguous amino acid sequence selected from HCV core proteins, or for diagnosing hepatitis C .

Hepatitis C includes various inflammatory diseases such as acute hepatitis, chronic hepatitis, liver cirrhosis, and liver cancer, which are caused by inflammatory diseases caused by infection with HCV. In addition, even if no clinical symptoms are present, it can be judged that hepatitis C has been infected if it is infected with HCV. The diagnosis of hepatitis C can be made by determining whether an individual is currently or previously infected with HCV, detecting an antigen or anti-HCV antibody in the individual, determining whether HCV is present in the individual, or detecting HCV in the individual May be inclusive.

The composition may be a liquid composition comprising the antibody in a liquid. The liquid may be one capable of dissolving and retaining the antibody. For example, water, or a buffer solution such as PBS. The composition may further comprise a substance that stably maintains the antibody.

The subject to be diagnosed may be a mammal. The mammal may be a human, a primate, a mouse, a rat, a cow, a pig, a horse, a sheep, a dog, a cat, or a combination thereof.

Another aspect is a pharmaceutical composition comprising a contiguous amino acid sequence selected from antibodies and non-structural protein 3 (NS3) that specifically binds to a polypeptide comprising a hydrophilic fragment selected from an HCV core protein and comprising a helicase activity An antigen of HCV comprising a fusion polypeptide comprising a C-terminal fragment of NS3, a hydrophilic fragment that is a contiguous amino acid sequence selected from a core protein, a hydrophilic fragment that is a contiguous amino acid sequence selected from NS4, and a contiguous amino acid sequence selected from NS5 And a composition for simultaneous detection of the antibody.

The fragment may be a whole protein or a sequence of consecutive amino acids selected from a peptide amino acid sequence.

Terminal domain comprises an amino acid sequence from amino acid 150 to C-terminal amino acid from N-terminus of NS3, amino acid sequence from amino acid 160 to C-terminal amino acid, amino acid sequence from amino acid 170 to C-terminal amino acid, An amino acid sequence from amino acid 180 to C-terminal amino acid, amino acid sequence from amino acid 185 to C-terminal amino acid, amino acid sequence from amino acid 160 to amino acid 620, or amino acid sequence from amino acid 170 to amino acid 600 . ≪ / RTI > For example, the HCV NS3 may be a C-terminal domain comprising the helicase activity of HCV NS3 consisting of the amino acid sequence of SEQ ID NO: 2.

The fusion polypeptide may be the C-terminal fragment of HCV NS3, the hydrophilic segment of the core protein, the hydrophilic segment of NS4, and the hydrophilic segment of NS5 linked by a bridge amino acid sequence or linker amino acid sequence. The bridging amino acid sequence or linker amino acid sequence may provide flexibility or rigidity to the fusion partner during fusion of the fusion partner and thus reduce the effect of each protein fragment in the fusion polypeptide on the protein structure of each other when folded or folded Lt; / RTI > In addition, the bridge amino acid sequence or linker amino acid sequence may contain a restriction element recognition sequence for facilitating the linkage of a nucleotide sequence encoding each protein. The bridge or linker amino acid sequence may comprise Gly, Asn and Ser residues, and may also include neutral amino acids such as Thr and Ala. Amino acid sequences suitable for peptide linkers are known in the art. On the other hand, the linker can variably determine its length within a range that does not affect the function of the fusion polypeptide. The bridge amino acid sequence or linker amino acid sequence may be a serine at both ends and a serine and glycine sequence with one, two, three, four, or five or more intervening glycines.

The bridge amino acid sequence or linker amino acid sequence may be inserted between the C-terminus of either fragment and the N-terminus of the other fragment subsequently linked. The bridge amino acid sequence or linker amino acid sequence may also be inserted between the C-terminus of the C-terminal fragment of NS3 and the N-terminus of the hydrophilic fragment of the core protein. The bridge amino acid sequence or linker amino acid sequence may also be inserted between the C-terminal of the hydrophilic fragment of the core protein and the N-terminal of the hydrophilic fragment of NS4. The bridge amino acid sequence or linker amino acid sequence may also be inserted between the C-terminal of the hydrophilic segment of NS4 and the N-terminus of the hydrophilic segment of NS5.

The fusion polypeptide comprises a C-terminal fragment of HCV NS3 comprising the amino acid sequence of SEQ ID NO: 2, a hydrophilic fragment of the core protein comprising the amino acid sequence of SEQ ID NO: 3, a hydrophilic fragment of NS4 comprising the amino acid sequence of SEQ ID NO: 4, A hydrophilic segment of NS5 comprising the amino acid sequence of SEQ ID NO: 5, and a bridge amino acid sequence comprising the amino acid sequence of SEQ ID NO: 6. The fusion polypeptide may also comprise the amino acid sequence of SEQ ID NO: 7, including both the C-terminal fragment of HCV NS3, the hydrophilic fragment of the core protein, the hydrophilic segment of NS4, the hydrophilic segment of NS5 and the bridge amino acid sequence. The nucleotide sequences encoding the amino acid sequences of SEQ ID NOS: 2 to 7 may be the nucleotide sequences of SEQ ID NOS: 9 to 18, respectively.

Since the fusion polypeptide can specifically bind to the anti-HCV antibody against the HCV antigen protein, the fusion polypeptide can be used to detect the anti-HCV antibody and diagnose the hepatitis C virus.

Among the terms or elements mentioned in the description of the composition, those already mentioned in the description of the polypeptide, antibody, hybridoma cell or polynucleotide are as described above.

Another aspect provides a kit for detecting an HCV antigen in an individual comprising an antibody or antigen-binding fragment thereof that specifically binds to a polypeptide comprising a hydrophilic fragment having a contiguous amino acid sequence selected from HCV core proteins.

The kit for detecting the HCV antigen can diagnose hepatitis C by measuring the amount of the HCV antigen as the hepatitis C diagnostic marker. The kit includes not only an antibody or an antigen-binding fragment thereof that specifically binds to a substance capable of measuring the amount of the HCV antigen described above, that is, an HCV antigen, but also an assay method for measuring the amount of the HCV antigen used by the kit Solution, or device suitable for use with the compositions of the invention.

The kit may comprise a substrate for immunological detection of an antigen, an antibody, an antigen-binding fragment, a polypeptide, a protein, or a secondary antibody and a secondary antibody labeled with a chromogenic or fluorescent substance that specifically binds to an HCV antigen, . The kit can measure the amount of HCV antigen by an ELISA method. ELISAs include direct ELISA using labeled antibodies that recognize the antigen attached to the solid support, indirect ELISA using labeled antibodies that recognize the capture antibody in a complex of antibodies recognizing the antigen attached to the solid support, A direct sandwich ELISA using another labeled antibody that recognizes an antigen in an antigen-antibody complex, an antibody that recognizes an antigen in a complex of an antigen and an antibody attached to a solid support, Indirect sandwich ELISA using a secondary antibody, and the like.

The kit comprising: a sample pad as a site to which a liquid sample containing an analyte is applied; a storage pad fluidly connected to the sample pad in a fluid-communicable manner, Is a membrane material that is a conjugate of protein A and gold particles and is in fluid communication with the storage pad and that moves the liquid sample by capillary movement, A chromatographic film material comprising a detection region that is non-diffusively immobilized; A hygroscopic pad in fluid communication with the chromatographic membrane material, and a solid support for supporting the sample pad, the storage pad, the chromatographic membrane material, and the hygroscopic pad. The protein A and the gold conjugate used in the kit of the present invention can be prepared by a method well known in the art. For example, a gold colloid solution may be prepared and gold in the solution may be combined with protein A.

Another aspect relates to the use of a nucleic acid sequence that is a contiguous amino acid sequence selected from NS3 and an antibody that specifically binds to a polypeptide comprising a hydrophilic fragment selected from an HCV core protein and which comprises a C terminal fragment of NS3 comprising helicase activity, There is provided a kit for simultaneous detection of an antigen of HCV and an antibody therefor comprising a fusion polypeptide comprising a hydrophilic fragment as an amino acid sequence, a hydrophilic fragment as a consecutive amino acid sequence selected from NS4 and a contiguous amino acid sequence selected from NS5 .

Among the terms or elements mentioned in the description of the kit, those already mentioned in the description of the polypeptide, antibody, hybridoma cell, polynucleotide or composition are as described above.

Another aspect relates to a method for detecting HCV infection comprising contacting a sample separated from an individual with an antibody that specifically binds to a polypeptide comprising a hydrophilic fragment selected from the HCV core protein to form a complex of the antibody with the HCV antigen contained in the sample; And measuring the level of the complex formed to measure the level of the HCV antigen in the sample. The present invention also provides a method for detecting an HCV antigen in a sample.

The antibody may bind to an HCV antigen to form a complex. By measuring the level of the complex formed, the level of HCV antigen in the sample can be measured.

Measuring the level of the complex may be accomplished by detecting a signal from a detectable label attached to a substance that specifically binds to the antibody or HCV antigen. Measuring the level of the complex may include determining the level of the antibody or HCV antigen by separating the complex again, measuring the level of a substance that specifically binds to the antibody or HCV antigen, Lt; RTI ID = 0.0 > HCV < / RTI >

The method may further comprise determining that the subject has been infected with HCV and has been afflicted with hepatitis C when the level of the measured HCV antigen is higher than the level of the same antigen measured in a normal control or negative control . In addition, the determining may further comprise determining that the subject is not infected with hepatitis C if the amount of the measured HCV antigen is less than or equal to the level of the same antigen measured in the normal or negative control . In addition, the method can also be used to determine whether the HCV antigen level of an individual is 1%, 2%, 3%, 4%, 5%, 10%, 20%, 30%, 40%, 50% Or more, more preferably 70%, 80%, 90%, 100%, 200%, 300%, 400%, 500%, 600%, 700%, 800%, 900% 5-fold, 10-fold, 20-fold, 30-fold, 50-fold, and 100-fold or more, it may be determined that the subject has hepatitis C infection.

Another aspect is a nucleic acid molecule that is a contiguous amino acid sequence selected from NS3 and an antibody that specifically binds to a polypeptide comprising a sample separated from an individual and a hydrophilic fragment selected from the HCV core protein and is a C-terminal fragment of NS3 comprising helicase activity, A hydrophilic fragment selected from the group consisting of a contiguous amino acid sequence selected from the group consisting of a protein, a protein, a continuous amino acid sequence selected from NS4, and a contiguous amino acid sequence selected from NS5 is contacted with a fusion polypeptide comprising the HCV antigen contained in the sample, Forming a first complex and a second complex of the fusion polypeptide with the anti-HCV antibody contained in the sample; And measuring the level of the first complex and the second complex formed to measure the level of the HCV antigen and the level of the anti-HCV antibody HBV antibody in the sample, respectively, to detect the HCV antigen and the anti-HCV antibody ≪ / RTI >

The fusion polypeptide can bind to a substance that can specifically bind to the contained in the sample to form a complex. Since the fusion polypeptide includes an HCV antigen, if an anti-HCV antibody is contained in the sample, it can specifically bind to the fusion polypeptide to form a complex.

Measuring the level of the complex may be accomplished by detecting a signal from a detectable label attached to a substance that specifically binds to the fusion polypeptide or anti-HCV antibody. Measuring the level of the complex may include determining the level of the fusion polypeptide or anti-HCV antibody, separating the complex, or measuring the level of a substance that specifically binds to the fusion polypeptide or anti-HCV antibody, And measuring the level of the fusion polypeptide or anti-HCV antibody without separating the fusion polypeptide or anti-HCV antibody.

The method may further comprise determining that the subject is afflicted with hepatitis C if the level of the anti-HCV antibody measured is higher than the level of the same antibody as measured in a normal or negative control. In addition, the determining may further comprise determining that the subject is not infected with hepatitis C if the amount of the anti-HCV antibody measured is equal to or lower than the level of the same antibody measured in a normal or negative control Lt; / RTI > The method also comprises administering to the subject an anti-HCV antibody level that is 1%, 2%, 3%, 4%, 5%, 10%, 20%, 30%, 40%, 50% , 70%, 80%, 90%, 100%, 200%, 300%, 400%, 500%, 600%, 700%, 800%, 900% , 5-fold, 10-fold, 20-fold, 30-fold, 50-fold, or 100-fold or more, the subject may be determined to have hepatitis C infection.

The measurements were performed using RT-PCR, competitive RT-PCR, real-time RT-PCR, RNase protection assay (RPA), Northern blotting, A nucleic acid microarray including DNA, Western blotting, enzyme linked immunosorbent assay (ELISA), radioimmunoassay (RIA), radioimmunodiffusion, Ouchterlony immunodiffusion, rocket immunization Immunoprecipitation assay, Complement Fixation Assay, FACS, mass spectrometry, magnetic bead-antibody immunoprecipitation, protein chip, or a combination thereof. . By measuring the level of the formed complex directly or indirectly, the HCV antigen or anti-HCV antibody contained in the sample can be detected to diagnose the presence or absence of hepatitis C in the individual.

The sample may be a biological sample derived from an individual. The biological sample may be blood, whole blood, serum, plasma, lymph fluid, urine, feces, tissue, cells, organs, bone marrow, saliva, sputum, cerebrospinal fluid or a combination thereof.

The method for detecting the HCV antigen and the method for detecting the anti-HCV antibody can be performed in parallel or sequentially.

Among the terms or elements mentioned in the description of the method, those already mentioned in the description of the polypeptide, antibody, hybridoma cell, polynucleotide, composition or kit are as described above.

An antibody that specifically binds to a polypeptide comprising a hydrophilic fragment of an HCV core protein according to an aspect and a fusion polypeptide comprising a C-terminal fragment of HCV NS3, a hydrophilic fragment of a core protein, a hydrophilic segment of NS4, and a hydrophilic segment of NS5 , It is possible to diagnose hepatitis C by detecting the HCV antigen and the antibody against the HCV antigen more easily and quickly.

FIG. 1 shows the result of testing HCV antigen and antibody-positive 15 samples with the HCV antigen antibody co-detection kit.
FIG. 2 shows the result of examining HCV antigen and 20 antibody-negative samples with the HCV antigen antibody co-detection kit.

Hereinafter, the present invention will be described in more detail with reference to examples. However, these examples are for illustrative purposes only, and the scope of the present invention is not limited to these examples.

Example  One. HCV  Recombination comprising core hydrophilic segments HCV  Antigen-assisted HCV  Preparation of Core Antibodies

One. HCV  Recombination comprising core hydrophilic segments HCV  Production of antigens

(One) HCV  Preparation of c160 antigen-expressing plasmid

The gene of the hydrophilic fragment of the HCV core protein was synthesized using the forward primer (SEQ ID NO: 19) and reverse primer (SEQ ID NO: 20) in Table 1 and the plasmid having Taq polymerase and HCV gene. Specifically, the DNA base sequence (SEQ ID NO: 8) of a site corresponding to 161 amino acids (SEQ ID NO: 1) from 2 amino acids of the HCV core gene was subjected to PCR synthesis. PCR was performed at 94 ° C for 1 min, at 55 ° C for 1 min, and at 72 ° C for 2 min for 1 cycle. The oligonucleotide primers used were 50 pmoles each. The amplified DNAs were separated by low melting agarose gel electrophoresis and digested with restriction enzymes BamHI (NEB R0136S, USA) and SalI (NEB, # R0138S, USA). Thereafter, recombinant expression plasmid pHCV c160 was constructed by linking the pGEX-4t expression plasmid treated with BamHI and SalI to the above DNAs.

[Table 1]

(2) HCV  Expression and purification of the antigen from the c160 antigen expression plasmid

The protein expression plasmid pHCV c160 was transformed into E. coli strain BL21. The transformed strain was cultured at 37 占 폚 in LB medium (1% peptone C, 0.5% yeast extract, 1% NaCl) containing 50 mg / ml of ampicillin, and when the absorbance (A600) -thio-D-galactoside) was added at a concentration of 1 mM and then cultured for another 16 hours. Cells were obtained from each culture medium at each time point. SDS polyacrylamide gel electrophoresis and Western blotting were performed on the cells, and the expression of HCV c160 antigen protein was confirmed. Then, the strain was cultured in a 37 ° C. fermentor for 2 hours using a 2 L culture medium, and IPTG was added at a concentration of 3 mM when the absorbance (A600) was 0.5. After addition of IPTG, culture was continued for 6 hours to induce HCV c160 protein expression. After completion of the culture, the culture was centrifuged to recover the cells. The recovered cells were washed twice with phosphate buffer (pH 7.4) and disrupted with an ultrasonic disintegrator. After centrifugation at 10,000 g for 30 minutes, only the soluble protein portion of the supernatant was separated. The supernatant containing the antigen was filtered with a 0.45 μm filter (Sartorius stedim, Ministart), and the filtrate was mixed with the equilibrium solution at a ratio of 1: 1. The mixed solution was entirely loaded in a glutathione-resin column and washed with an equilibrium solution until OD ₂₈₀ = 0.05 or less. The recombinant HCV c160 antigen was then recovered from the eluate.

2. HCV  Preparation of Core 160 antibody

(One) HCV  Manufacture of Core 160 Antigen Immune Mouse

Six-week old female BALB / c mice were immunized with purified HCV c160 antigen as an immunogen. Specifically, the mice were intraperitoneally injected four times at an interval of one week at an amount of 200 μL / mouse. One week after the last intraperitoneal injection, the same antigen was diluted in phosphate buffered saline (PBS) at a concentration of 20% (v / v) and injected into the tail vein three times (20 μl / s) at 1 day intervals. After the 3 rd intravenous injection, a midtrigger test was performed with serum obtained from the tail vein. As a result of the test for the potency, a mouse in which the amount of the antibody was sufficiently obtained was selected.

(2) HCV  Core antibody Hybridoma  Cell line production

The splenocytes of the selected mice were detached and filtered through a cell strainer (BD falcon) having a size of 70 mu m pore size. The filtered spleen cells were centrifuged to obtain precipitates, and 5 ml of red blood cell lysis buffer (Sigma) was added thereto, followed by allowing to stand for 1 minute to remove red blood cells. Spleen cells from which red cells were removed were washed three times with DMEM (Dulbecco's Modified Eagle's Medium, Gibco) and cell counts were performed. The splenocytes were mixed with a mouse derived myeloma cell line SP2 / 0-Ag 14 (ATCC CRL-1581) to a mixing ratio of 10: 1 (splenocytes: SP2 / 0). Mixed cells were washed twice with DMEM and fused using PEG 1500 (Roche). Cell fusion was performed with PEG1500 spiked, and after the fusion was completed, the precipitate was obtained by centrifugation. (HAT medium) supplemented with 10% of HAT (Gibco), antibiotics (Gibco) and fetal bovine serum (Hyclone) (hereinafter referred to as "HAT medium") was added to a 96-well tissue culture plate at a volume of 200 μl / And then grown in an incubator for 3 days.

(3) Hybridoma  Cell line culture and selection of fusion cell line

The fusion cell lines were grown in a cell culture incubator maintained at 37 ° C, 5% carbon dioxide gas and humidification conditions, and fused cell lines were selected by replacing the HAT medium for one week at 2-day intervals. Cell lines that had been screened with HAT medium were screened for positive clones by enzyme-linked immunosorbent assay (ELISA). First, the fused cell culture medium growing in each well of a 96-well tissue culture plate was dispensed into a 96-well adsorption plate (Costar) coated with the HCV core antigen at a concentration of 2.5 μg / ml in a volume of 100 μl / And reacted for 1 hour. After 1 hour of reaction, the cells were washed five times, and anti-mouse IgG HRP conjugate and anti-mouse IgM HRP conjugate were dispensed at a volume of 100 μl / well and reacted at 37 ° C for 30 minutes. After 30 minutes of reaction, the plate was washed five times, and the substrate solution was dispensed at a volume of 100 쨉 l / well, followed by color development for 15 minutes. Finally, the reaction stop solution was added in a volume of 100 l / well. After stopping the reaction, absorbance was measured at a wavelength of 450 nm in the reader, and positive clone mAb anti-HCV c160 # 11 and mAb anti-HCV c160 # 75 showing high values were selected. The selected positive clones were transferred to a 24-well tissue culture plate, cultured for 3 days, and secondary screened in the same manner as above. Hybridoma cell lines obtained through secondary screening were subjected to limiting dilution to select the final single positive clone candidates HCV c160 # 11, HCV c160 # 75. The final selected monoclonal antibody-producing hybridomas were designated as mAb anti-HCV c160 (# 11), mAb anti-HCV c160 (# 75) and deposited with the Korean Cell Line Research Foundation (KCLRF) . The hybridoma cells prepared according to the above examples were deposited with the Korean Cell Line Research Foundation under the Budapest Treaty on Dec. 10, 2015 (accession numbers: # 11 KCLRF-BP-00354 and # 75 KCLRF-BP -00355). On the other hand, the deposited Hybridoma cells are stored in accordance with the provisions of the Budapest Treaty on the Deposit of Microorganisms and can be distributed to the general public with reference to the above accession numbers.

(4) The anti- HCV  Production and purification of c160 antibody

The final positive clones were transferred to a T75 (SPL) culture dish, cultured for 3 days, injected into the abdominal cavity of each mouse at a density of 1 × 10 ⁶ cells / ml at a density of 1 × 10 ⁶ cells / ml, and collected a week later. The mouse multiple solutions were centrifuged (3000 rpm, 10 minutes, 4 ° C) to precipitate the supernatant, and the supernatant was mixed with binding buffer (Thermo scientific) at a ratio of 1: 1, followed by reaction at room temperature for 1 hour . After 1 hour, the suspension was centrifuged again (3000 rpm, 10 minutes, 4 ° C) to precipitate the suspension, and the non-precipitated suspension was filtered out again using a 1.2 μm pore size filter. The aspirate was completely removed and placed in a column containing protein G resin (Pierce) to induce binding between antibody and protein G. The multiple solutions were passed through the column, washed three times with phosphate buffer, and then eluted with an elution buffer (Thermo Scientific). The eluted antibody was again applied to a PD-10 column (GE heathcare) and eluted with a phosphate buffer solution for final purification.

Example  2. HCV  For antibody detection HCV NS3  Preparation of Core 45 Fusion Antigens Potency  black

One. HCV  Preparation of NS3 core 45 fusion gene recombinant antigen

(1) Preparation of HCV NS3 core 45 fusion antigen expression plasmid

(SEQ ID NO: 9) having a helicase activity of HCV NS3, a hydrophilic fragment (SEQ ID NO: 10) of a core protein, a hydrophilic fragment of NS4 (SEQ ID NO: 11) and a hydrophilic fragment of NS5 Using synthetic PCR to generate HCV NS3 core 45 plasmid DNA (SEQ ID NO: 18). Serine + glycine bridge sequences (SEQ ID NOS: 13, 14, 15, 16 or 17) were added between the respective regions so that each antigen site was not tangled when folded after purification.

Specifically, synthetic PCR was performed using primers and Taq polymerase shown in Table 2 below. The PCR was carried out at 94 ° C for 1 min, at 55 ° C for 1 min, and at 72 ° C for 2 min. The reaction was repeated 30 times. The concentration of oligonucleotides used was 50 pmole. The amplified DNAs were separated by low melting agarose gel electrophoresis and digested with restriction enzymes BamHI (NEB R0136S, USA) and SalI (NEB, # R0138S, USA). Then, recombinant expression plasmid pHCV NS3 core 45 DNA was prepared by linking the pGEX-4t expression plasmid treated with BamHI and SalI to the above DNAs. 3 as a hydrophilic fragment of the core protein, SEQ ID NO: 4 as a hydrophilic segment of NS4, SEQ ID NO: 5 as a hydrophilic segment of NS5, and a bridge amino acid sequence of SEQ ID NO: 5 as a hydrophilic segment of NS5 as a hydrophilic segment of NS3 helicase, The fusion peptide of SEQ ID NO: 7 including all of the amino acid sequence of SEQ ID NO: 6 is expressed.

[Table 2]

(2) HCV  Expression and purification of antigen from NS3 core 45 fusion antigen expression plasmid

Protein expression plasmid pHCV NS3 core 45 was transformed into E. coli strain BL21. The transformed strain was cultured at 37 占 폚 in LB medium (1% peptone C, 0.5% yeast extract, 1% NaCl) containing 5 ug / ml of ampicillin, and when the absorbance (A600) -thio-D-galactoside) was added at a concentration of 1 mM and then cultured for another 16 hours. Cells were obtained from each culture in each hour, SDS polyacrylamide gel electrophoresis and Western blotting were carried out on the cells, and the expression of HCV NS3 core 45 fusion antigen protein was confirmed. Then, the strain was cultured in a 37 ° C. fermentor for 2 hours using a 2 L culture medium, and IPTG was added at a concentration of 3 mM when the absorbance (A600) was 0.5. After the addition of IPTG, the culture was continued for 6 hours to induce HCV NS3 core 45 fusion antigen protein expression. After completion of the culture, the culture was centrifuged to recover the cells. The recovered cells were washed twice with phosphate buffer (pH 7.4) and disrupted with an ultrasonic disintegrator. After centrifugation at 10,000 g for 30 minutes, only the soluble protein portion of the supernatant was separated. The supernatant containing the antigen was filtered with a 0.45 μm filter (Sartorius stedim, Ministart), and the filtrate was mixed with the equilibrium solution at a ratio of 1: 1. The mixed solution was entirely loaded in a glutathione-resin column and washed with an equilibrium solution until OD ₂₈₀ = 0.05 or less. The recombinant HCV NS3 core 45 fusion antigen was then recovered from the eluate.

2. HCV  NS3 core 45 antigen Potency  black

The purified HCV NS3 core 45 fusion antigen was identified by enzyme-linked immunosorbent assay (ELISA) using a HBsAg mixed titer Accususet Performance Panel (0805-0217, Seracure).

The antigen was coated on a 96-well adsorption plate (Costar) at a concentration of 2.5 μg / ml. Samples were dispensed at a volume of 10 μl / well and reacted at 37 ° C. for 1 hour. After 1 hour of incubation, the cells were washed five times, and the human IgG HRP conjugate was dispensed at a volume of 100 μl / well, followed by reaction at 37 ° C. for 30 minutes. After 30 minutes of reaction, the plate was washed five times, and the substrate solution was dispensed at a volume of 100 μl / well to perform a color reaction for 15 minutes. Finally, the reaction stop solution was added in a volume of 100 μl / well. After stopping the reaction, absorbance was measured at a wavelength of 450 nm in the reader. The results are shown in Table 3 below.

[Table 3] HCV  NS3 core 45 antigen Potency  Test result

Example  3. HCV  Antigen and Antibody to It Diagnostic Kit  Manufacturing and effect verification

One. Kit  Produce

(One) Inspection line  Produce

Two test lines were used to prepare the HCV antigen and antibody detection kit of the present preparation. In the first test line, mAb anti-HCV c160 (# 11) was quantitated by absorbance and BCA method, and diluted to a concentration of 2 mg / mL. Diluted mAB anti-HCV c160 (# 11) was dispensed onto the first test line.

In the second test line, the purified HCV NS3 core 45 fusion antigen was quantified by absorbance and BCA method as in Example 1, and then diluted to a concentration of 2 mg / mL. The diluted HCV NS3 core 45 fusion antigen was dispensed onto the second test line. The prepared test lines were kept in airtight after drying and used in the experiment.

(2) Inspection line  Label manufacture

As a label for the first test, a gold-conjugated single clone anti-HCV c160 (# 75) was used. Specifically, gold chloride was dissolved in distilled water at 0.01%, and the mixture was heated for about 10 minutes while adding 0.1% sodium citrate solution and then cooled. The prepared colloidal gold solution was refrigerated. Then, the monoclonal anti-HCV c160 (# 75) was dissolved in physiological saline to a final concentration of 20 μg / ml, and the colloidal gold solution was reacted for 10 minutes to induce a ligation reaction. The gold particles were stabilized and centrifuged at 10,000 g for 30 minutes to form a precipitate. The resulting precipitate was dissolved in physiological saline, filtered through a 0.45 탆 filter, and then absorbance was measured at 540 nm and diluted to a final value of 20. As a label for the second inspection, gold was attached to the HCV NS3 core 45 fusion antigen in the same manner as the labeling method for the first inspection line.

2. HCV  Antigen and Antibody Concurrent Of the diagnostic kit  Check the effect

The prepared HCV antigen and antibody simultaneous diagnostic kit was tested as follows.

(1) Use samples

① Hepatitis C antibody positive samples: HCV mixed titer Accuset Performance Panel (0810-0175, Seracure) 16 samples were purchased and used. Sixteen specimens were used as positive specimens.

② Hepatitis C antigen-positive specimen: HBV, HCV, HIV antibody-negative HBV and HCV antigen-negative serum samples obtained from Bundang CHA Hospital were added to 15 samples among 20 serum samples and added with 1 ~ 10 μL of recombinant antigen c160 And a hepatitis C antigen positive specimen was prepared.

③ Hepatitis C antigen / antibody negative specimen: Twenty serum samples of HBV, HCV, HIV antibody negative and HBV and HCV antigen negative samples obtained from Bundang Cha Hospital were used.

(2) Inspection method

10 μL of the sample (blood or serum) was dropped into the sample inlet, and then 90 to 100 μL of the test diluent was added. After 15 minutes, read the red purple band that appeared in the test window, and when the contrast medium was developed, the negative, control and test line 1 positive for hepatitis C antigen for color development, positive for control line and test line 2, positive for hepatitis C antibody, 1 and test line 2 were judged to be positive for hepatitis C antigens and antibodies.

(3) Results

As shown in Fig. 1, color development was observed in both test lines 1 and 2 in all 15 samples with hepatitis C antigen / antibody concurrently. Also, as shown in Fig. 2, only the color development of the control line was observed in all 20 samples of the hepatitis C antigen / antibody negative.

[Agency]

Depositor Name: Korea Cell Line Research Foundation

Accession number: KCLRFBP00354

Funding date: 20151210

Depositor Name: Korea Cell Line Research Foundation

Accession number: KCLRFBP00355

Funding date: 20151210

<110> SBIinternational <120> A composition, kit and method for concurrent detecting          Hepatitis C virus antigen and antibody <130> PN112312 <160> 28 <170> Kopatentin 2.0 <210> 1 <211> 160 <212> PRT <213> Hepatitis C Virus <400> 1 Ser Thr Asn Pro Lys Pro Gln Arg Lys Thr Lys Arg Asn Thr Asn Arg   1 5 10 15 Arg Pro Gln Asp Val Lys Phe Pro Gly Gly Gly Gln Ile Val Gly Gly              20 25 30 Val Tyr Leu Leu Pro Arg Arg Gly Pro Arg Leu Gly Val Arg Ala Thr          35 40 45 Arg Lys Thr Ser Glu Arg Ser Gln Pro Arg Gly Arg Arg Gln Pro Ile      50 55 60 Pro Lys Ala Arg Arg Pro Glu Gly Arg Thr Trp Ala Gln Pro Gly Tyr  65 70 75 80 Pro Trp Pro Leu Tyr Gly Asn Gly Gly Met Gly Trp Ala Gly Trp Leu                  85 90 95 Leu Ser Pro Arg Gly Ser Arg Pro Ser Trp Gly Pro Thr Asp Pro Arg             100 105 110 Arg Arg Ser Ser Asn Leu Gly Lys Val Ile Asp Thr Leu Thr Cys Gly         115 120 125 Phe Ala Asp Leu Met Gly Tyr Ile Pro Leu Val Gly Ala Pro Leu Gly     130 135 140 Gly Ala Ala Arg Ala Leu Ala His Gly Val Arg Val Leu Glu Asp Gly 145 150 155 160 <210> 2 <211> 433 <212> PRT <213> Hepatitis C Virus <400> 2 Thr Asp Ser Ser Pro Pro Ala Val Pro Gln Thr Phe Gln Val Ala   1 5 10 15 His Leu His Ala Pro Thr Gly Ser Gly Lys Ser Thr Lys Val Pro Ala              20 25 30 Ala Tyr Ala Gln Gly Tyr Lys Val Leu Val Leu Asn Pro Ser Val          35 40 45 Ala Ala Thr Leu Gly Phe Gly Ala Tyr Met Ser Lys Ala Tyr Gly Thr      50 55 60 Asp Pro Asn Val Arg Thr Gly Val Arg Thr Ile Thr Thr Gly Ser Pro  65 70 75 80 Ile Thr Tyr Ser Thr Tyr Gly Lys Phe Leu Ala Asp Gly Gly Cys Ser                  85 90 95 Gly Gly Ala Tyr Asp Ile Ile Ile Cys Asp Glu Cys His Ser Thr Asp             100 105 110 Ser Thr Thr Ile Leu Gly Ile Gly Thr Val Leu Asp Gln Ala Glu Thr         115 120 125 Ala Gly Ala Arg Leu Val Val Leu Ala Thr Ala Thr Pro Pro Gly Ser     130 135 140 Val Thr Val Pro His Pro Asn Ile Glu Glu Val Gly Leu Ser Asn Thr 145 150 155 160 Gly Glu Ile Pro Phe Tyr Gly Lys Ala Ile Pro Ile Glu Val Ile Lys                 165 170 175 Gly Gly Arg His Leu Ile Phe Cys His Ser Lys Lys Lys Cys Asp Glu             180 185 190 Leu Ala Ala Lys Leu Ser Ala Leu Gly Ile Asn Ala Val Ala Tyr Tyr         195 200 205 Arg Gly Leu Asp Val Ser Val Ile Pro Ala Ser Gly Asp Val Val Val     210 215 220 Val Ala Thr Asp Ala Leu Met Thr Gly Phe Thr Gly Asp Phe Asp Ser 225 230 235 240 Val Ile Asp Cys Asn Thr Cys Val Thr Gln Thr Val Asp Phe Ser Leu                 245 250 255 Asp Pro Thr Phe Thr Ile Glu Thr Thr Thr Val Gln Asp Ala Val             260 265 270 Ser Arg Ser Gln Arg Arg Gly Arg Thr Gly Arg Gly Arg Ala Gly Ile         275 280 285 Tyr Arg Phe Val Thr Pro Gly Glu Arg Pro Ser Gly Met Phe Asp Ser     290 295 300 Ser Val Leu Cys Glu Cys Tyr Asp Ser Gly Cys Ala Trp Tyr Glu Leu 305 310 315 320 Thr Pro Ala Glu Thr Ser Val Arg Leu Arg Ala Tyr Leu Asn Thr Pro                 325 330 335 Gly Leu Pro Val Cys Gln Asp His Leu Glu Phe Trp Glu Ser Val Phe             340 345 350 Thr Gly Leu Thr His Ile Asp Ala His Phe Leu Ser Gln Thr Lys Gln         355 360 365 Ala Gly Asp Asn Phe Pro Tyr Leu Val Ala Tyr Gln Ala Thr Val Cys     370 375 380 Ala Arg Ser Gln Ala Pro Pro Pro Ser Trp Asp Gln Met Trp Lys Cys 385 390 395 400 Leu Ile Arg Leu Lys Pro Thr Leu His Gly Pro Thr Pro Leu Leu Tyr                 405 410 415 Arg Leu Gly Ala Val Gln Asn Glu Val Thr Leu Thr His Pro Ile Thr             420 425 430 Lys     <210> 3 <211> 20 <212> PRT <213> Hepatitis C Virus <400> 3 Ser Thr Asn Pro Lys Pro Gln Arg Lys Thr Lys Arg Asn Thr Asn Arg   1 5 10 15 Arg Pro Gln Asp              20 <210> 4 <211> 18 <212> PRT <213> Hepatitis C Virus <400> 4 Ser Arg Gly Asn His Val Ser Pro Thr His Tyr Val Pro Glu Ser Asp   1 5 10 15 Ala Ala         <210> 5 <211> 20 <212> PRT <213> Hepatitis C Virus <400> 5 Pro Ile Trp Ala Arg Pro Asp Tyr Asn Pro Pro Leu Leu Glu Ser Trp   1 5 10 15 Lys Asp Pro Asp              20 <210> 6 <211> 5 <212> PRT <213> Artificial Sequence <220> <223> serine + glycine bridge <400> 6 Ser Gly Gly Gly Ser   1 5 <210> 7 <211> 506 <212> PRT <213> Artificial Sequence <220> <223> HCV NS3 core 45 protein seq <400> 7 Thr Asp Ser Ser Pro Pro Ala Val Pro Gln Thr Phe Gln Val Ala   1 5 10 15 His Leu His Ala Pro Thr Gly Ser Gly Lys Ser Thr Lys Val Pro Ala              20 25 30 Ala Tyr Ala Gln Gly Tyr Lys Val Leu Val Leu Asn Pro Ser Val          35 40 45 Ala Ala Thr Leu Gly Phe Gly Ala Tyr Met Ser Lys Ala Tyr Gly Thr      50 55 60 Asp Pro Asn Val Arg Thr Gly Val Arg Thr Ile Thr Thr Gly Ser Pro  65 70 75 80 Ile Thr Tyr Ser Thr Tyr Gly Lys Phe Leu Ala Asp Gly Gly Cys Ser                  85 90 95 Gly Gly Ala Tyr Asp Ile Ile Ile Cys Asp Glu Cys His Ser Thr Asp             100 105 110 Ser Thr Thr Ile Leu Gly Ile Gly Thr Val Leu Asp Gln Ala Glu Thr         115 120 125 Ala Gly Ala Arg Leu Val Val Leu Ala Thr Ala Thr Pro Pro Gly Ser     130 135 140 Val Thr Val Pro His Pro Asn Ile Glu Glu Val Gly Leu Ser Asn Thr 145 150 155 160 Gly Glu Ile Pro Phe Tyr Gly Lys Ala Ile Pro Ile Glu Val Ile Lys                 165 170 175 Gly Gly Arg His Leu Ile Phe Cys His Ser Lys Lys Lys Cys Asp Glu             180 185 190 Leu Ala Ala Lys Leu Ser Ala Leu Gly Ile Asn Ala Val Ala Tyr Tyr         195 200 205 Arg Gly Leu Asp Val Ser Val Ile Pro Ala Ser Gly Asp Val Val Val     210 215 220 Val Ala Thr Asp Ala Leu Met Thr Gly Phe Thr Gly Asp Phe Asp Ser 225 230 235 240 Val Ile Asp Cys Asn Thr Cys Val Thr Gln Thr Val Asp Phe Ser Leu                 245 250 255 Asp Pro Thr Phe Thr Ile Glu Thr Thr Thr Val Gln Asp Ala Val             260 265 270 Ser Arg Ser Gln Arg Arg Gly Arg Thr Gly Arg Gly Arg Ala Gly Ile         275 280 285 Tyr Arg Phe Val Thr Pro Gly Glu Arg Pro Ser Gly Met Phe Asp Ser     290 295 300 Ser Val Leu Cys Glu Cys Tyr Asp Ser Gly Cys Ala Trp Tyr Glu Leu 305 310 315 320 Thr Pro Ala Glu Thr Ser Val Arg Leu Arg Ala Tyr Leu Asn Thr Pro                 325 330 335 Gly Leu Pro Val Cys Gln Asp His Leu Glu Phe Trp Glu Ser Val Phe             340 345 350 Thr Gly Leu Thr His Ile Asp Ala His Phe Leu Ser Gln Thr Lys Gln         355 360 365 Ala Gly Asp Asn Phe Pro Tyr Leu Val Ala Tyr Gln Ala Thr Val Cys     370 375 380 Ala Arg Ser Gln Ala Pro Pro Pro Ser Trp Asp Gln Met Trp Lys Cys 385 390 395 400 Leu Ile Arg Leu Lys Pro Thr Leu His Gly Pro Thr Pro Leu Leu Tyr                 405 410 415 Arg Leu Gly Ala Val Gln Asn Glu Val Thr Leu Thr His Pro Ile Thr             420 425 430 Lys Ser Gly Gly Gly Ser Ser Thr Asn Pro Lys Pro Gln Arg Lys Thr         435 440 445 Lys Arg Asn Thr Asn Arg Arg Pro Gln Asp Ser Gly Gly Gly Ser Ser     450 455 460 Arg Gly Asn His Val Ser Pro Thr His Tyr Val Pro Glu Ser Asp Ala 465 470 475 480 Ala Ser Gly Gly Gly Ser Pro Ile Trp Ala Arg Pro Asp Tyr Asn Pro                 485 490 495 Pro Leu Leu Glu Ser Trp Lys Asp Pro Asp             500 505 <210> 8 <211> 480 <212> DNA <213> Hepatitis C Virus <400> 8 agcacgaatc ctaaacctca aagaaaaacc aaacgtaaca ccaaccgccg cccacaggac 60 gtcaagtttc cgggcggtgg tcagatcgtt ggtggagttt acctgttgcc gcgcaggggc 120 cccaggttgg gtgtgcgcgc gactaggaag acttccgagc ggtcgcaacc tcgcggaagg 180 cgacaaccta tccccaaggc tcgccggccc gagggcagga cctgggctca gcccgggtac 240 ccttggcccc tctatggcaa tgagggcatg gggtgggcag gatggctcct gtcaccccgc 300 ggctctcggc ctagttgggg ccccacggac ccccggcgta ggtcgcgcaa tttgggtaag 360 gtcatcgata ccctcacatg cggcttcgcc gacctcatgg ggtacattcc gctcgtcggc 420 gcccccttag ggggcgctgc cagggccctg gcacatggtg tccgggttct ggaggacggc 480                                                                          480 <210> 9 <211> 1299 <212> DNA <213> Hepatitis C Virus <400> 9 acagacaact cctccccccc ggcagtaccg cagacattcc aagtggccca tctacacgct 60 cccaccggca gtggcaagag caccaaagtg ccagctgcgt acgcagccca agggtacaag 120 gtactagtcc tgaacccgtc tgttgccgcg accttaggtt ttggggcgta tatgtccaag 180 gcatatggta ccgaccccaa tgtcagaact ggggtaagga ccatcaccac gggcagcccc 240 atcacgtact ccacctacgg caagttcctt gctgacggcg gttgctctgg gggcgcctac 300 gacatcataa tatgcgatga gtgccactca actgactcga ctaccatctt gggcatcggc 360 acagtcctgg accaagcgga gacggctgga gcgcggctcg tcgtgctcgc caccgctaca 420 cctccggggt cggtcaccgt gccacaccca aatatcgagg aagtgggcct gtccaatact 480 ggggagatcc ccttctatgg caaagccatc cccatcgagg tcatcaaggg gggaaggcat 540 ctcatcttct gccattccaa gaagaagtgt gacgagctcg ccgcgaagct gtcggccctc 600 ggaatcaatg ctgtagcata ttaccggggt cttgatgtgt ccgtcatacc ggctagcgga 660 gacgtcgttg tcgtggcaac agacgcccta atgacgggct tcaccggcga ctttgactca 720 gtcatcgact gtaacacatg tgtcacccag acagtcgatt tcagcttgga ccccaccttc 780 accattgaga cgacgaccgt gccccaagac gcggtgtcgc gctcgcagcg gcgaggcagg 840 actggtaggg gtagggcagg catctacagg tttgtgactc cgggagaacg gccctcgggc 900 atgttcgatt cctcggtcct gtgtgagtgt tatgactcgg gctgtgcttg gtacgaactc 960 acgcccgctg agacctcggt taggttgcgg gcatacctaa atacaccagg gctgcccgtc 1020 tgtcaggacc atctggagtt ctgggagagt gtcttcacag gcctcaccca catagatgcc 1080 cacttcttgt cccaaactaa gcaggcagga gacaacttcc cctacctggt agcataccaa 1140 gctacagtgt gcgccaggtc tcaggcccca cctccatcgt gggatcaaat gtggaagtgt 1200 ctcatacggc tcaagcccac actgcacggg ccaacgcccc tgctgtatag gctaggagcc 1260 gttcaaaatg aggtcaccct cacacacccc ataaccaaa 1299 <210> 10 <211> 60 <212> DNA <213> Hepatitis C Virus <400> 10 agcacgaatc ctaaacctca aagaaaaacc aaacgtaaca ccaaccgccg cccacaggac 60                                                                           60 <210> 11 <211> 54 <212> DNA <213> Hepatitis C Virus <400> 11 tcgcggggta accacgtctc ccccacgcac tatgtgcctg agagcgacgc agca 54 <210> 12 <211> 60 <212> DNA <213> Hepatitis C Virus <400> 12 cccatatggg cacgcccgga ttacaatcct cccctgctag agtcctggaa ggatccggac 60                                                                           60 <210> 13 <211> 15 <212> DNA <213> Artificial Sequence <220> <223> serine + glycine bridge <400> 13 tctggagggg gttcc 15 <210> 14 <211> 15 <212> DNA <213> Artificial Sequence <220> <223> serine + glycine bridge <400> 14 agcggggggg ggagc 15 <210> 15 <211> 15 <212> DNA <213> Artificial Sequence <220> <223> serine + glycine bridge <400> 15 agcggtggtg gcagt 15 <210> 16 <211> 15 <212> DNA <213> Artificial Sequence <220> <223> serine + glycine bridge <400> 16 tccgggggtg gttca 15 <210> 17 <211> 15 <212> DNA <213> Artificial Sequence <220> <223> serine + glycine bridge <400> 17 tcaggtggcg ggtcg 15 <210> 18 <211> 1533 <212> DNA <213> Artificial Sequence <220> <223> HCV NS3 core 45 DNA seq <400> 18 ggatccacag acaactcctc ccccccggca gtaccgcaga cattccaagt ggcccatcta 60 cacgctccca ccggcagtgg caagagcacc aaagtgccag ctgcgtacgc agcccaaggg 120 tacaaggtac tagtcctgaa cccgtctgtt gccgcgacct taggttttgg ggcgtatatg 180 tccaaggcat atggtaccga ccccaatgtc agaactgggg taaggaccat caccacgggc 240 agccccatca cgtactccac ctacggcaag ttccttgctg acggcggttg ctctgggggc 300 gcctacgaca tcataatatg cgatgagtgc cactcaactg actcgactac catcttgggc 360 atcggcacag tcctggacca agcggagacg gctggagcgc ggctcgtcgt gctcgccacc 420 gctacacctc cggggtcggt caccgtgcca cacccaaata tcgaggaagt gggcctgtcc 480 aatactgggg agatcccctt ctatggcaaa gccatcccca tcgaggtcat caagggggga 540 aggcatctca tcttctgcca ttccaagaag aagtgtgacg agctcgccgc gaagctgtcg 600 gccctcggaa tcaatgctgt agcatattac cggggtcttg atgtgtccgt cataccggct 660 agcggagacg tcgttgtcgt ggcaacagac gccctaatga cgggcttcac cggcgacttt 720 gactcagtca tcgactgtaa cacatgtgtc acccagacag tcgatttcag cttggacccc 780 accttcacca ttgagacgac gaccgtgccc caagacgcgg tgtcgcgctc gcagcggcga 840 ggcaggactg gtaggggtag ggcaggcatc tacaggtttg tgactccggg agaacggccc 900 tcgggcatgt tcgattcctc ggtcctgtgt gagtgttatg actcgggctg tgcttggtac 960 gaactcacgc ccgctgagac ctcggttagg ttgcgggcat acctaaatac accagggctg 1020 cccgtctgtc aggaccatct ggagttctgg gagagtgtct tcacaggcct cacccacata 1080 gatgcccact tcttgtccca aactaagcag gcaggagaca acttccccta cctggtagca 1140 taccaagcta cagtgtgcgc caggtctcag gccccacctc catcgtggga tcaaatgtgg 1200 aagtgtctca tacggctcaa gcccacactg cacgggccaa cgcccctgct gtataggcta 1260 ggagccgttc aaaatgaggt caccctcaca caccccataa ccaaatctgg agggggttcc 1320 agcacgaatc ctaaacctca aagaaaaacc aaacgtaaca ccaaccgccg cccacaggac 1380 tctggagggg gttcctcgcg gggtaaccac gtctccccca cgcactatgt gcctgagagc 1440 gacgcagcat ctggaggggg ttcccccata tgggcacgcc cggattacaa tcctcccctg 1500 ctagagtcct ggaaggatcc ggactaagtc gac 1533 <210> 19 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> HCV c160 Forward primer <400> 19 ggatccagca cgaatcctaa acct 24 <210> 20 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> HCV c160 Reverse primer <400> 20 gtcgacttag ccgtcctcca gaacccg 27 <210> 21 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> NS3 forward primer <400> 21 ggatccacag acaactcctc cccc 24 <210> 22 <211> 33 <212> DNA <213> Artificial Sequence <220> <223> NS3 reverse primer <400> 22 ggaaccccct ccagatttgg ttatggggtg tgt 33 <210> 23 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> core forward primer <400> 23 ggatccagca cgaatcctaa acct 24 <210> 24 <211> 75 <212> DNA <213> Artificial Sequence <220> <223> core reverse primer <400> 24 gtcctgtggg cggcggttgg tgttacgttt ggtttttctt tgaggtttag gattcgtgct 60 ggaaccccct ccaga 75 <210> 25 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> NS4 forward primer <400> 25 ggatcctcgc ggggtaacca cgtc 24 <210> 26 <211> 69 <212> DNA <213> Artificial Sequence <220> <223> NS4 reverse primer <400> 26 tgctgcgtcg ctctcaggca catagtgcgt gggggagacg tggttacccc gcgaggaacc 60 ccctccaga 69 <210> 27 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> NS5 forward primer <400> 27 ggatccccca tatgggcacg cccg 24 <210> 28 <211> 75 <212> DNA <213> Artificial Sequence <220> <223> NS5 reverse primer <400> 28 gtccggatcc ttccaggact ctagcagggg aggattgtaa tccgggcgtg cccatatggg 60 ggaaccccct ccaga 75

Claims (15)

Wherein the polypeptide comprises a hydrophilic fragment that is a contiguous amino acid sequence selected from the hepatitis C virus (HCV) core protein. The polypeptide according to claim 1, wherein the hydrophilic fragment having a contiguous amino acid sequence selected from the HCV core protein comprises the amino acid sequence of SEQ ID NO: 1. An antibody or an antigen-binding fragment thereof which is produced in a hybridoma cell, which is KCLRF-BP-00354 or KCLRF-BP-00355, and which specifically binds to the polypeptide of claim 1. Wherein the accession number is KCLRF-BP-00354 or KCLRF-BP-00355. A polynucleotide encoding the polypeptide of claim 1. A composition for detecting an antigen of HCV comprising an antibody or an antigen-binding fragment thereof that specifically binds to the polypeptide of claim 1. A kit for detecting an antigen of HCV comprising an antibody that specifically binds to the polypeptide of claim 1. A continuous amino acid sequence selected from an antibody and non-structural protein 3 (NS3) that specifically binds to the polypeptide of claim 1 and a C-terminal fragment of NS3 comprising helicase activity, Wherein the fusion polypeptide comprises a hydrophilic fragment that is a contiguous amino acid sequence selected from NS4 and NS5, and a hydrophilic fragment that is a contiguous amino acid sequence selected from NS4 and NS5, and an antibody thereto. 9. The composition of claim 8, wherein the fusion polypeptide comprises at least one amino acid sequence selected from the group consisting of SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4 and SEQ ID NO: 9. The composition of claim 8, wherein the fusion polypeptide comprises all of the amino acid sequences of SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4 and SEQ ID NO: A continuous amino acid sequence selected from an antibody and non-structural protein 3 (NS3) that specifically binds to the polypeptide of claim 1 and a C-terminal fragment of NS3 comprising helicase activity, A kit for the simultaneous detection of an antigen of HCV and an anti-HCV antibody comprising a fusion polypeptide comprising a hydrophilic fragment as an amino acid sequence, a hydrophilic fragment as a contiguous amino acid sequence selected from NS4, and a contiguous amino acid sequence selected from NS5. In order to provide information necessary for diagnosis of hepatitis C in an individual,
Contacting a sample separated from the subject with an antibody that specifically binds to the polypeptide of claim 1 to form a complex of the antibody with the HCV antigen contained in the sample; And
Measuring the level of the complex formed and measuring the level of HCV antigen in the sample. 13. The method of claim 12, further comprising determining that the subject is afflicted with hepatitis C if the measured level of HCV antigen is higher than the level of the same antigen as measured in a normal control. In order to provide information necessary for diagnosis of hepatitis C in an individual,
A continuous amino acid sequence selected from an antibody that specifically binds to the polypeptide of claim 1 and a non-structural protein 3 and a C-terminal fragment of NS3 comprising a helicase activity, a contiguous amino acid sequence selected from a core protein , A hydrophilic fragment that is a contiguous amino acid sequence selected from NS4 and a hydrophilic fragment that is a contiguous amino acid sequence selected from NS5, and a first complex of the antibody and a sample contained therein RTI ID = 0.0 &gt; HCV &lt; / RTI &gt; antibody and the fusion polypeptide; And
Measuring the level of the first complex and the second complex formed to measure the level of the HCV antigen and the level of the anti-HCV antibody HBV antibody in the sample, respectively, and detecting the HCV antigen and the anti-HCV antibody in the sample . 15. The method of claim 14, wherein when the level of the measured HCV antigen is higher than the level of the same antigen measured in the normal control and the level of the measured anti-HCV antibody is higher than the level of the same antibody measured in the normal control, Determining that the patient is afflicted with hepatitis.

Images