KR101784455B1 - Fusion polypeptide comprising hydrophilic fragments of Hepatitis C virus, composition, kit and method for diagnosing Hepatitis C virus infection comprising the same - Google Patents

Abstract

A fusion polypeptide comprising a hydrophilic fragment of hepatitis C virus, a composition, a kit, and a method for diagnosing hepatitis C virus infection including the fusion polypeptide.

Description

TECHNICAL FIELD [0001] The present invention relates to a fusion polypeptide comprising a hydrophilic fragment of hepatitis C virus, a composition, a kit, and a method for diagnosing hepatitis C virus infection including the same, C virus infection comprising the same}

A fusion polypeptide comprising a hydrophilic fragment of hepatitis C virus, a composition, a kit, and a method for diagnosing hepatitis C virus infection including the fusion polypeptide.

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 antibodies against hepatitis C virus is required more easily and quickly.

One aspect is to provide a separate fusion polypeptide comprising three or more hydrophilic fragments that are contiguous amino acid sequences selected from the amino acid sequence of hepatitis C virus non-structural protein 3 (HCV NS3).

Another aspect is to provide an isolated polynucleotide encoding said fusion polypeptide.

Another aspect is to provide a kit and a composition for diagnosing hepatitis C virus infection comprising said fusion polypeptide.

Another aspect is to provide a method of diagnosing hepatitis C virus infection comprising contacting a sample isolated from an individual with said fusion polypeptide.

One aspect provides a separate fusion polypeptide comprising three or more hydrophilic fragments that are contiguous amino acid sequences selected from the amino acid sequence of hepatitis C virus non-structural protein 3 (HCV NS3).

The HCV NS3 has two structural domains, for example, an N-terminal domain conferred by activated serine protease activity belonging to the maturation of the viral protein, and a helicase associated with NTPase activity that plays a role in the replication of the viral genome helicase < / RTI > activity.

In one embodiment, the HCV NS3 may comprise a C-terminal domain comprising a helicase activity. The C-terminal domain comprising the helicase activity includes 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 C- 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 170 Lt; / RTI > to 600 amino acids. 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: 1.

As used herein, the term "hydrophilic fragment" may refer to a region having a hydrophilic character including a contiguous amino acid sequence, such as the Kyte-Doolittle method (Kyte, J. and Doolittle, , J. Mol. Bio., 157, 105-132). The hydrophilic segment is partially hydrophilic and only a portion of the amino acid residues are capable of interacting with water, and the entire portion of the segment need not be hydrophilic. For example, if the amino acid sequence of the fragment contains a polar amino acid including lysine, arginine, histidine, aspartic acid, serine, threonine, glutamic acid, asparagine, glutamine, cysteine, glycine and proline, the fragment may have hydrophilicity , And even if the amino acid sequence of the fragment contains a hydrophobic amino acid, it may not lose hydrophilicity. Even if the fragment is composed of a hydrophobic amino acid, it can be a region having hydrophilicity due to the influence of the adjacent hydrophilic amino acid.

In one embodiment, the hydrophilic fragment may comprise the amino acid sequence of SEQ ID NO: 3, SEQ ID NO: 5, or SEQ ID NO: 7. Thus, the fusion polypeptide according to one embodiment may comprise the amino acid sequence of SEQ ID NO: 3, SEQ ID NO: 5 and SEQ ID NO: 7.

Each of the at least three hydrophilic fragments may be linked by a linker (e. G., A peptide linker).

As used herein, the term "linker" refers to a molecule that binds two different fusion partners (e. G., Hydrophilic fragments) to hydrogen bonding, electrostatic interactions, van der Waals forces, disulfide bonds, salt bridges, hydrophobic interactions, And may be connected to each other by means of coupling or the like. The linker may have at least one cysteine capable of participating in at least one disulfide bond under physiological conditions or other standard polypeptide conditions (e. G., Polypeptide purification conditions, polypeptide storage conditions) , It may also serve as a hinge to provide a gap of a certain size between the fusion partners or to provide flexibility or rigidity to the fusion body. The linker may be a peptide linker, for example, a linker consisting of a plurality of amino acids. The imager linker may be composed of, for example, 1 to 100, 2 to 100, or 2 to 50 amino acids.

In one embodiment, the peptide linker 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. The peptide linker may be linked to the C-terminus of one fusion fragment or to the N-terminus of another fusion fragment. On the other hand, the peptide linker can determine its length in various ways as long as it does not affect the function of the fusion polypeptide.

In another embodiment, the peptide linker may comprise the amino acid sequence of SEQ ID NO: 25.

In one embodiment, the fusion polypeptide may be composed of the amino acid sequence of SEQ ID NO: The amino acid sequence of SEQ ID NO: 15 includes the hydrophilic fragment of SEQ ID NO: 3, the hydrophilic fragment of SEQ ID NO: 5, and the hydrophilic fragment of SEQ ID NO: 7, and each hydrophilic fragment may be linked by the amino acid sequence of SEQ ID NO: .

The amino acid sequence of the fusion polypeptide can be suitably modified to the extent that it does not substantially change the target 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.

Another aspect is an isolated polynucleotide encoding a fusion polypeptide comprising at least three hydrophilic fragments from the C-terminal domain of hepatitis C virus non-structural protein 3 (HCV NS3) to provide.

The HCV NSC or its C-terminal domain, hydrophilic fragment, and fusion polypeptide are as described above.

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 not only a perfectly complementary sequence but also a substantially complementary sequence, which can be used under stringent conditions known in the art, for example, a nucleotide encoding the amino acid sequence of the fusion polypeptide May refer to a sequence that can hybridize with a nucleotide sequence of a sequence.

In addition, the nucleotide sequence encoding the amino acid sequence of the fusion 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 fusion polypeptide may also 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.

In one embodiment, the nucleotide sequence encoding the C-terminal domain of HCV NS3 may be comprised of the nucleotide sequence of SEQ ID NO: 2. Further, the nucleotide sequence encoding the hydrophilic fragment may be composed of the nucleotide sequence of SEQ ID NO: 4, SEQ ID NO: 6, or SEQ ID NO: Thus, a polynucleotide encoding a fusion polypeptide according to one embodiment may comprise a nucleotide sequence of SEQ ID NO: 4, SEQ ID NO: 6, and SEQ ID NO: 8.

In one embodiment, the polynucleotide encoding the fusion polypeptide may further comprise a nucleotide sequence encoding a peptide linker for linking the at least three hydrophilic fragments. For example, the nucleotide sequence encoding the peptide linker may comprise one or more nucleotide sequences selected from the group consisting of SEQ ID NOS: 26 to 30.

In one embodiment, the polynucleotide encoding the fusion polypeptide may be the nucleotide sequence of SEQ ID NO: 16. Wherein the nucleotide sequence of SEQ ID NO: 16 comprises the nucleotide sequence of SEQ ID NO: 4, the nucleotide sequence of SEQ ID NO: 6, and the nucleotide sequence of SEQ ID NO: 8, wherein each nucleotide sequence is selected from the group consisting of SEQ ID NOs: 26 to 30 Or may comprise one or more nucleotide sequences.

The preparation of the fusion 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 aforementioned fusion polypeptide is inserted, transforming the host cell with the recombinant expression vector, culturing the transformed host cell, And recovering the fusion polypeptide expressed in the host cell, wherein the fusion polypeptide comprises at least three hydrophilic fragments from the hepatitis C virus non-structural protein 3 or its C-terminal domain.

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, and the foreign DNA fragment may be a polynucleotide encoding the aforementioned fusion 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.

The fusion polypeptide expressed by the host cell may be secreted extracellularly by the secretory signal peptide, in which case it can be obtained by recovering it from the culture broth or medium. For example, the culture supernatant can be concentrated using a commercially available protein filter to isolate the fusion polypeptide. However, when expressed without the secretory signal sequence, the fusion polypeptide can be obtained directly from the cell lysate since it is in the intracellular periplasmic space. Processes for isolating fused 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, fusion polypeptides obtained from such cultures may be further purified by methods known in the art. For example, depending on the fusion polypeptide to be recovered, the fusion polypeptide may be purified by chromatography (e.g., ion exchange, affinity, hydrophobicity and size exclusion), chromatofocusing, SDS-PAGE, fractional dissolution ≪ RTI ID = 0.0 > phat precipitation). ≪ / RTI >

Another aspect provides a composition for diagnosing hepatitis C virus infection comprising a separate fusion polypeptide comprising at least three hydrophilic fragments from hepatitis C virus non-structural protein 3 or its C-terminal domain.

The HCV NSC or its C-terminal domain, the hydrophilic fragment, and the fusion polypeptide, and polynucleotides, vectors, and host cells for preparing the same are as described above.

As used herein, the term "diagnosis of hepatitis C virus infection" is used to determine whether an individual is currently or previously infected with the hepatitis C virus, determine whether an antibody against hepatitis C virus is present in the individual, Detection of an antibody against hepatitis virus, determination of the presence of hepatitis C virus in an individual, or detection of hepatitis C virus in an individual. The diagnosis of the hepatitis C virus infection may also include the diagnosis of a disease caused by hepatitis C virus infection, such as hepatitis (e. G., Hepatitis C), or a diagnosis of lymphoma.

In the above composition or kit, the fusion polypeptide may be alone or in combination with another substance. The other material may be a label capable of generating or generating a detectable signal. The term "detectable label" is an atom or molecule that specifically detects a molecule containing a label, among molecules of the same kind without a label, and the detectable label is, for example, colored bead, an antigenic crystal, an enzyme, a hybridizable nucleic acid, a chromogenic material, a fluorescent material, a phosphorescent material, an electrically detectable molecule, a molecule or quantum dot that provides altered fluorescence-polarization or altered light-diffusion. The label may also include radioactive isotopes such as P ³² and S ³⁵ , chemiluminescent compounds, labeled binding proteins, spectroscopic markers such as heavy metal atoms and dyes, and magnetic labels.

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

The kit may comprise a substance that binds to the antibody and / or a second antibody. For example, a labeled or unlabeled material, an anti-human antibody, an anti-rabbit antibody, or an anti-mouse antibody. In addition, the kit may further comprise reagents necessary for an assay reaction using an antibody such as ELISA or immunofluorescence analysis.

The kit includes a sample pad to which a sample is applied; A conjugate pad fluidly connected to the sample pad and having a substance capable of binding to the hepatitis C virus antibody movably supported; A chromatographic material in fluid communication with the conjugate pad and moving the sample, the chromatographic material comprising at least three hydrophilic fragments from hepatitis C virus non-structural protein 3 or its C-terminal domain A chromatography membrane material comprising a detection region in which the fusion polypeptide 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 chromatography membrane material, and the moisture absorption pad.

Another aspect is a method for detecting a hepatitis C virus comprising contacting a sample isolated from an individual with a separate fusion polypeptide comprising at least three hydrophilic fragments from hepatitis C virus non-structural protein 3 or its C-terminal domain; And

A method for detecting hepatitis C virus antibody for providing information on the diagnosis of hepatitis C virus, comprising the step of detecting the presence of a complex of the fusion polypeptide and the hepatitis C virus antibody in the sample, The method comprising:

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 subject may be a mammal, such as a human, cow, horse, pig, dog, sheep, goat, or cat.

The contacting step may be static or dynamic in the liquid medium. The contacting may be, for example, on a kit as described above.

The step of detecting the presence of the complex may be accomplished by methods known in the art. For example, by detecting a signal from a detectable label attached to a substance that specifically binds to the fusion polypeptide or anti-HCV antibody. The step of detecting the presence of the complex may include separating the complex and determining the level of the fusion polypeptide or anti-HCV antibody, 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 complex. 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, it is possible to detect the presence or absence of hepatitis C in the individual by detecting the anti-HCV antibody contained in the sample.

In addition, the method may further comprise, when a complex is detected, determining that the subject is a hepatitis C virus infection.

In one embodiment, the fusion polypeptide may be one that binds to an antibody in a subject having hepatitis C virus infection. The fusion polypeptide has significant antigenicity compared to a fusion polypeptide in which one hydrophilic fragment or two hydrophilic fragments from HCV NS3 or its C-terminal domain is fused, and has a specific binding ability to an antibody in an individual having hepatitis C virus infection . Accordingly, fusion polypeptides, compositions and kits thereof, according to one embodiment, may be usefully used to diagnose hepatitis C virus infection.

The composition, kit, and method for diagnosing hepatitis C virus infection comprising the fusion polypeptide according to one aspect of the present invention can detect the antibody against the hepatitis C virus in a more simple and accurate manner.

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 NS3 Preparation of a fusion polypeptide comprising a hydrophilic fragment of hepatitis C virus non-structural protein 3 and its antigenicity and stability evaluation

One. HCV NS3 Of a fusion polypeptide comprising a hydrophilic fragment of

(1.1) HCV NS3 Of a fusion polypeptide expression plasmid comprising the hydrophilic fragment of

In order to prepare a fusion polypeptide containing a hydrophilic fragment of HCV NS3, the DNA of Korean HCV (isolate: C_59-12 / south korea) was used as template DNA and an expression plasmid for each fragment was prepared.

First, as the control fragment, the C-terminal domain of HCV NS3 (hereinafter referred to as 'NS3f1') having the amino acid sequence shown in SEQ ID NO: 1 was used.

In order to prepare a fusion polypeptide comprising the hydrophilic fragment of HCV NS3, three hydrophilic fragments of the hydrophilic fragment present in the HCV NS3 gene, specifically a fragment having amino acids 1 to 84 from the N terminus of SEQ ID NO: 1 (SEQ ID NO: 3 (SEQ ID NO: 5, hereinafter referred to as 'NS3f3') and fragments having the amino acids 261 to 396 (SEQ ID NO: 7, hereinafter referred to as 'NS3f4' , And combinations thereof. Hereinafter, the fusion polypeptide (SEQ ID NO: 9) of NS3f2 and NS3f3 is referred to as 'NS3f5', the fusion polypeptide of NS3f3 and NS3f4 (SEQ ID NO: 11) is referred to as 'NS3f6', the fusion polypeptide of NS3f2 and NS3f4 NS3f7 ', and the fusion polypeptide of NS3f2, NS3f3, and NS3f4 (SEQ ID NO: 15) is referred to as NS3f8.

In order to synthesize the respective fragments, PCR (Polymerase Chain Reaction) was performed using the primers shown in Table 1 and Taq polymerase. PCR was performed at 94 ° C for 1 minute, at 55 ° C for 1 min, and at 72 ° C for 2 min for 1 cycle. The oligonucleotide primers used were 50 pmoles each.

primer SEQ ID NO: NS3f1 forward primer SEQ ID NO: 17 NS3f1 reverse primer SEQ ID NO: 18 NS3f2 forward primer SEQ ID NO: 19 NS3f2 reverse primer SEQ ID NO: 20 NS3f3 forward primer SEQ ID NO: 21 NS3f3 Reverse primer SEQ ID NO: 22 NS3f4 forward primer SEQ ID NO: 23 NS3f4 reverse primer SEQ ID NO: 24

The fusion polypeptide antigens of NS3f5, NS3f6, NS3f7, and NS3f8 are further linked by serine and glycine bridges (amino acid sequence of SEQ ID NO: 25, nucleic acid sequence of SEQ ID NO: 26 to SEQ ID NO: 30) between the respective fragments, After refining, each piece was prevented from tangling by folding.

The amplified DNA fragments were separated by low melting agarose gel electrophoresis and cut with restriction enzymes BamHI (NEB # R0136S, USA) and SalI (NEB, # R0138S, USA) . In addition, the pGEX-4t expression plasmid was digested with restriction enzymes BamHI (NEB # R0136S, USA) and SalI (NEB, # R0138S, USA). Next, the DNA fragments and the plasmid were ligated to each other to prepare recombinant expression plasmids pHCV NS3f1 to NS3f8 DNA.

(1.2) HCV NS3 Lt; RTI ID = 0.0 > of a < / RTI > hydrophilic fragment of the fusion polypeptide

In order to purify the fragment of HCV NS3 prepared above, the protein expression plasmid thus prepared was transformed into E. coli strain BL21. The thus transformed strains were incubated at 37 ° C in LB medium (1% pectin, 0.5% yeast extract, 1% NaCl) containing 50 g / ml of ampicillin and the absorbance (A600) -D-galactoside) was added at a concentration of 1 mM and cultured for 16 hours. The culture broth was taken every hour, and the cell lysate was subjected to SDS polyacrylamide gel electrophoresis and Western blotting. As a result, it was confirmed that the HCV NS3 fragment was expressed.

Then, the transformed strain BL21 was cultured in a 2 L culture medium for 2 hours at 37 ° C. in a fermenter, and added with 3 mM of IPTG when the absorbance was 0.5 (A600). The cells were further cultured for 6 hours to induce the expression of the antigen fragments. After the culture was completed, the culture broth was centrifuged to collect the cells, 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. Thereafter, the supernatant of the antigen-containing 0.45 ㎛ filter (Sartorius stedim, Ministart) equilibrium solution (NaN3 / sigma, PBS (NaCl, KCl, Na ₂ HPO the supernatant antigen is contained was filtered to _4, KH ₂ PO ₄ ) / sigma, ionized water) at a ratio of 1: 1. Next, all the antigens were loaded on a glutathione resin column (GE healthcare), and then washed with an equilibrium solution until the OD ₂₈₀ became 0.05 or less. The eluate (1 M Tris (pH 8.0) / sigma, L-Glutathione reduced / sigma (G6529-25G)). The recovered antigen fragments were quantified using BCA method (Pierce, USA) and stored at -70 ° C until use.

2. HCV NS3 Of the fusion polypeptide comprising the hydrophilic fragment of

(2.1) Evaluation of antigenicity

Enzyme-linked immunosorbent assay (ELISA) was performed to evaluate the antigenicity of the purified 8 kinds of antigen fragments.

Specifically, the purified HCV NS3 antigen fragment was coated on a 96-well adsorption plate (Costar) at a concentration of 2.5 ug / ml. Subsequently, a sample of HCV mixed titer Accuset Performance Panel (0810-0175, Seracure) was dispensed at a volume of 10 μl / well and reacted at 37 ° C for 1 hour. After 1 hour of reaction, the cells were washed five times, and an anti-human IgG HRP conjugate (A0170, Sigma) was 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 tetramethylbenzidine (kem-en-tec) was dispensed at a rate of 100 μl / well and color reaction was performed for 15 minutes. 1N sulfuric acid (Junsei) Was added. After stopping the reaction, absorbance was measured at a wavelength of 450 nm in a reader (sunrise, tecan), and the results are shown in Table 2 below.

NS3f1 NS3f2 NS3f3 NS3f4 NS3f5 NS3f6 NS3f7 NS3f8 Panel number Absorbance value Absorbance value Absorbance value Absorbance value Absorbance value Absorbance value Absorbance value Absorbance value One 1.854 0.521 0.431 1.754 0.741 1.945 1.865 2.014 2 2.031 0.753 0.638 1.264 0.659 1.784 1.650 2.954 3 2.214 0.625 0.715 1.954 0.815 2.305 2.018 2.654 4 2.069 0.485 0.628 1.531 0.664 1.954 1.867 2.982 5 1.846 0.512 0.465 1.623 0.718 1.608 1.594 2.654 6 1.659 0.451 0.543 1.532 0.603 1.840 1.765 2.715 7 2.068 0.745 0.642 1.846 0.794 1.952 1.846 2.935 8 2.155 0.632 0.522 1.478 0.805 2.064 1.996 2.748 9 2.096 0.516 0.717 1.654 0.669 1.841 1.695 2.953 10 2.134 0.534 0.425 1.669 0.674 1.854 1.749 2.965 11 1.406 0.231 0.302 1.039 0.457 1.156 1.148 2.159 12 1.550 0.217 0.419 1.265 0.534 1.628 1.526 2.064 13 2.165 0.468 0.549 1.642 0.650 1.745 1.648 2.953 14 2.220 0.521 0.556 1.488 0.649 1.655 1.526 2.784 15 2.046 0.625 0.472 1.674 0.708 1.950 1.864 2.685 16 0.041 0.023 0.033 0.041 0.031 0.036 0.033 0.043

As shown in Table 2 above, the HCV NS3 antigen comprising one or two hydrophilic fragments was not highly antigenic compared to the control group, but the fusion polypeptide antigen comprising three HCV NS3 hydrophilic fragments according to one embodiment, Or an HCV NS3 antigen comprising two hydrophilic fragments.

(2.2) Evaluation of stability

To evaluate the stability of the antigen fragments, the NS3f8 antigen and the control antigen NS3f1 antigen were stored at 4 DEG C, -20 DEG C, and -40 DEG C for 12 months. Then, the antigenicity was evaluated in the same manner as in Example (2.1) above, and the results are shown in Table 3 below.

NS3f1 NS3f8 Immediately after manufacture 12 months storage Immediately after manufacture 12 months storage 4 ℃ -20 ° C -40 ° C 4 ℃ -20 ° C -40 ° C Panel number Absorbance value Absorbance value Absorbance value Absorbance value Absorbance value Absorbance value Absorbance value Absorbance value One 1.847 0.954 1.522 1.784 2.140 2.051 2.210 2.105 2 2.003 1.124 1.856 2.065 2.947 2.794 2.915 2.867 3 2.156 1.023 2.014 2.231 2.784 2.655 2.718 2.651 4 1.984 0.954 1.568 2.084 2.843 2.947 2.864 2.915 5 1.748 0.815 1.547 1.654 2.719 2.594 2.648 2.715 6 1.954 0.944 1.748 1.847 2.695 2.614 2.694 2.765 7 2.164 0.748 1.945 2.064 2.975 2.855 2.905 2.933 8 2.064 1.360 1.845 1.985 2.840 2.748 2.695 2.718 9 2.114 0.965 2.136 2.084 2.916 2.965 2.903 2.846 10 1.987 0.822 1.749 2.031 3.0 < 2.849 2.914 3.0 < 11 1.531 0.465 1.598 1.627 2.213 2.105 2.047 2.164 12 1.624 0.815 1.824 1.718 1.984 2.014 2.140 2.157 13 2.031 1.007 1.965 2.063 3.0 < 2.894 2.966 3.0 < 14 2.147 0.925 2.065 2.214 2.964 2.746 2.823 2.814 15 2.061 0.834 1.763 1.903 2.745 2.641 2.632 2.651 16 0.042 0.033 0.047 0.034 0.042 0.043 0.044 0.041

As shown in Table 3, it can be confirmed that the antigenicity of the control antigens was remarkably decreased after 12 months storage regardless of the storage temperature. In contrast, the fusion polypep- tide antigen comprising three HCV NS3 hydrophilic fragments according to one embodiment shows little or no change in antigenicity after 12 months storage regardless of storage temperature.

As a result, the fusion polypeptide antigen according to one embodiment is excellent in antigenicity and stability and can be used for diagnosis of hepatitis C virus.

<110> SBIinternational <120> Fusion polypeptides containing hydrophilic fragments of Hepatitis          C virus, composition, kit and method for diagnosing Hepatitis C          virus infection comprising the same <130> PN112453 <160> 30 <170> Kopatentin 2.0 <210> 1 <211> 433 <212> PRT <213> Hepatitis C virus fragment 1 <400> 1 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> 2 <211> 1299 <212> DNA <213> Hepatitis C virus fragment 1 <400> 2 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> 3 <211> 85 <212> PRT <213> Hepatitis C virus fragment 2 <400> 3 Phe Thr Asp Ser Ser Pro Pro Ala Val Pro Gln Thr Phe Gln Val   1 5 10 15 Ala His Leu His Ala Pro Thr Gly Ser Gly Lys Ser Thr Lys Val Pro              20 25 30 Ala Ala Tyr Ala Ala Gln Gly Tyr Lys Val Leu Val Leu Asn Pro Ser          35 40 45 Val Ala Ala Thr Leu Gly Phe Gly Ala Tyr Met Ser Lys Ala Tyr Gly      50 55 60 Thr Asp Pro Asn Val Arg Thr Gly Val Arg Thr Ile Thr Thr Gly Ser  65 70 75 80 Pro Ile Thr Tyr Ser                  85 <210> 4 <211> 255 <212> DNA <213> Hepatitis C virus fragment 2 <400> 4 ttcacagaca actcctcccc cccggcagta ccgcagacat tccaagtggc ccatctacac 60 gctcccaccg gcagtggcaa gagcaccaaa gtgccagctg cgtacgcagc ccaagggtac 120 gt; aaggcatatg gtaccgaccc caatgtcaga actggggtaa ggaccatcac cacgggcagc 240 cccatcacgt actcc 255 <210> 5 <211> 72 <212> PRT <213> Hepatitis C virus fragment 3 <400> 5 Gly Gly Cys Ser Gly Gly Ala Tyr Asp Ile Ile Ile Cys Asp Glu Cys   1 5 10 15 His Ser Thr Asp Ser Thr Thr Ile Leu Gly Ile Gly Thr Val Leu Asp              20 25 30 Gln Ala Glu Thr Ala Gly Ala Arg Leu Val Val Leu Ala Thr Ala Thr          35 40 45 Pro Pro Gly Ser Val Thr Val Pro His Pro Asn Ile Glu Glu Val Gly      50 55 60 Leu Ser Asn Thr Gly Glu Ile Pro  65 70 <210> 6 <211> 216 <212> DNA <213> Hepatitis C virus fragment 3 <400> 6 ggcggttgct ctgggggcgc ctacgacatc ataatatgcg atgagtgcca ctcaactgac 60 tcgactacca tcttgggcat cggcacagtc ctggaccaag cggagacggc tggagcgcgg 120 ctcgtcgtgc tcgccaccgc tacacctccg gggtcggtca ccgtgccaca cccaaatatc 180 gaggaagtgg gcctgtccaa tactggggag atcccc 216 <210> 7 <211> 136 <212> PRT <213> Hepatitis C virus fragment 4 <400> 7 Thr Ile Glu Thr Thr Thr Val Gln Asp Ala Val Ser Ser Ser Gln   1 5 10 15 Arg Arg Gly Arg Thr Gly Arg Gly Arg Ala Gly Ile Tyr Arg Phe Val              20 25 30 Thr Pro Gly Glu Arg Pro Ser Gly Met Phe Asp Ser Ser Val Leu Cys          35 40 45 Glu Cys Tyr Asp Ser Gly Cys Ala Trp Tyr Glu Leu Thr Pro Ala Glu      50 55 60 Thr Ser Val Arg Leu Arg Ala Tyr Leu Asn Thr Pro Gly Leu Pro Val  65 70 75 80 Cys Gln Asp His Leu Glu Phe Trp Glu Ser Val Phe Thr Gly Leu Thr                  85 90 95 His Ile Asp Ala His Phe Leu Ser Gln Thr Lys Gln Ala Gly Asp Asn             100 105 110 Phe Pro Tyr Leu Val Ala Tyr Gln Ala Thr Val Cys Ala Arg Ser Gln         115 120 125 Ala Pro Pro Pro Ser Trp Asp Gln     130 135 <210> 8 <211> 408 <212> DNA <213> Hepatitis C virus fragment 4 <400> 8 accattgaga cgacgaccgt gccccaagac gcggtgtcgc gctcgcagcg gcgaggcagg 60 actggtaggg gtagggcagg catctacagg tttgtgactc cgggagaacg gccctcgggc 120 atgttcgatt cctcggtcct gtgtgagtgt tatgactcgg gctgtgcttg gtacgaactc 180 acgcccgctg agacctcggt taggttgcgg gcatacctaa atacaccagg gctgcccgtc 240 tgtcaggacc atctggagtt ctgggagagt gtcttcacag gcctcaccca catagatgcc 300 cacttcttgt cccaaactaa gcaggcagga gacaacttcc cctacctggt agcataccaa 360 gctacagtgt gcgccaggtc tcaggcccca cctccatcgt gggatcaa 408 <210> 9 <211> 162 <212> PRT <213> Hepatitis C virus fragment 5 <400> 9 Phe Thr Asp Ser Ser Pro Pro Ala Val Pro Gln Thr Phe Gln Val   1 5 10 15 Ala His Leu His Ala Pro Thr Gly Ser Gly Lys Ser Thr Lys Val Pro              20 25 30 Ala Ala Tyr Ala Ala Gln Gly Tyr Lys Val Leu Val Leu Asn Pro Ser          35 40 45 Val Ala Ala Thr Leu Gly Phe Gly Ala Tyr Met Ser Lys Ala Tyr Gly      50 55 60 Thr Asp Pro Asn Val Arg Thr Gly Val Arg Thr Ile Thr Thr Gly Ser  65 70 75 80 Pro Ile Thr Ser Ser Gly Gly Gly Ser Gly Gly Cys Ser Gly Gly                  85 90 95 Ala Tyr Asp Ile Ile Ile Cys Asp Glu Cys His Ser Thr Asp Ser Thr             100 105 110 Thr Ile Leu Gly Ile Gly Thr Val Leu Asp Gln Ala Glu Thr Ala Gly         115 120 125 Ala Arg Leu Val Val Leu Ala Thr Ala Thr Pro Pro Gly Ser Val Thr     130 135 140 Val Pro His Pro Asn Ile Glu Glu Val Gly Leu Ser Asn Thr Gly Glu 145 150 155 160 Ile Pro         <210> 10 <211> 486 <212> DNA <213> Hepatitis C virus fragment 5 <400> 10 ttcacagaca actcctcccc cccggcagta ccgcagacat tccaagtggc ccatctacac 60 gctcccaccg gcagtggcaa gagcaccaaa gtgccagctg cgtacgcagc ccaagggtac 120 gt; aaggcatatg gtaccgaccc caatgtcaga actggggtaa ggaccatcac cacgggcagc 240 cccatcacgt actcctctgg agggggttcc ggcggttgct ctgggggcgc ctacgacatc 300 ataatatgcg atgagtgcca ctcaactgac tcgactacca tcttgggcat cggcacagtc 360 ctggaccaag cggagacggc tggagcgcgg ctcgtcgtgc tcgccaccgc tacacctccg 420 gggtcggtca ccgtgccaca cccaaatatc gaggaagtgg gcctgtccaa tactggggag 480 atcccc 486 <210> 11 <211> 213 <212> PRT <213> Hepatitis C virus fragment 6 <400> 11 Gly Gly Cys Ser Gly Gly Ala Tyr Asp Ile Ile Ile Cys Asp Glu Cys   1 5 10 15 His Ser Thr Asp Ser Thr Thr Ile Leu Gly Ile Gly Thr Val Leu Asp              20 25 30 Gln Ala Glu Thr Ala Gly Ala Arg Leu Val Val Leu Ala Thr Ala Thr          35 40 45 Pro Pro Gly Ser Val Thr Val Pro His Pro Asn Ile Glu Glu Val Gly      50 55 60 Leu Ser Asn Thr Gly Glu Ile Pro Ser Gly Gly Gly Ser Thr Ile Glu  65 70 75 80 Thr Thr Val Pro Gln Asp Ala Val Ser Ser Ser Gln Arg Arg Gly                  85 90 95 Arg Thr Gly Arg Gly Arg Ala Gly Ile Tyr Arg Phe Val Thr Pro Gly             100 105 110 Glu Arg Pro Ser Gly Met Phe Asp Ser Ser Val Leu Cys Glu Cys Tyr         115 120 125 Asp Ser Gly Cys Ala Trp Tyr Glu Leu Thr Pro Ala Glu Thr Ser Val     130 135 140 Arg Leu Arg Ala Tyr Leu Asn Thr Pro Gly Leu Pro Val Cys Gln Asp 145 150 155 160 His Leu Glu Phe Trp Glu Ser Val Phe Thr Gly Leu Thr His Ile Asp                 165 170 175 Ala His Phe Leu Ser Gln Thr Lys Gln Ala Gly Asp Asn Phe Pro Tyr             180 185 190 Leu Val Ala Tyr Gln Ala Thr Val Cys Ala Arg Ser Gln Ala Pro Pro         195 200 205 Pro Ser Trp Asp Gln     210 <210> 12 <211> 639 <212> DNA <213> Hepatitis C virus fragment 6 <400> 12 ggcggttgct ctgggggcgc ctacgacatc ataatatgcg atgagtgcca ctcaactgac 60 tcgactacca tcttgggcat cggcacagtc ctggaccaag cggagacggc tggagcgcgg 120 ctcgtcgtgc tcgccaccgc tacacctccg gggtcggtca ccgtgccaca cccaaatatc 180 gaggaagtgg gcctgtccaa tactggggag atcccctctg gagggggttc caccattgag 240 acgacgaccg tgccccaaga cgcggtgtcg cgctcgcagc ggcgaggcag gactggtagg 300 ggtagggcag gcatctacag gtttgtgact ccgggagaac ggccctcggg catgttcgat 360 tcctcggtcc tgtgtgagtg ttatgactcg ggctgtgctt ggtacgaact cacgcccgct 420 gagacctcgg ttaggttgcg ggcataccta aatacaccag ggctgcccgt ctgtcaggac 480 catctggagt tctggagag tgtcttcaca ggcctcaccc acatagatgc ccacttcttg 540 tcccaaacta agcaggcagg agacaacttc ccctacctgg tagcatacca agctacagtg 600 tgcgccaggt ctcaggcccc acctccatcg tgggatcaa 639 <210> 13 <211> 226 <212> PRT <213> Hepatitis C virus fragment 7 <400> 13 Phe Thr Asp Ser Ser Pro Pro Ala Val Pro Gln Thr Phe Gln Val   1 5 10 15 Ala His Leu His Ala Pro Thr Gly Ser Gly Lys Ser Thr Lys Val Pro              20 25 30 Ala Ala Tyr Ala Ala Gln Gly Tyr Lys Val Leu Val Leu Asn Pro Ser          35 40 45 Val Ala Ala Thr Leu Gly Phe Gly Ala Tyr Met Ser Lys Ala Tyr Gly      50 55 60 Thr Asp Pro Asn Val Arg Thr Gly Val Arg Thr Ile Thr Thr Gly Ser  65 70 75 80 Pro Ile Thr Ser Ser Gly Gly Ser Ser Thr Ile Glu Thr Thr Thr                  85 90 95 Val Pro Gln Asp Ala Val Ser Arg Ser Gln Arg Arg Gly Arg Thr Gly             100 105 110 Arg Gly Arg Ala Gly Ile Tyr Arg Phe Val Thr Pro Gly Glu Arg Pro         115 120 125 Ser Gly Met Phe Asp Ser Ser Val Leu Cys Glu Cys Tyr Asp Ser Gly     130 135 140 Cys Ala Trp Tyr Glu Leu Thr Pro Ala Glu Thr Ser Val Arg Leu Arg 145 150 155 160 Ala Tyr Leu Asn Thr Pro Gly Leu Pro Val Cys Gln Asp His Leu Glu                 165 170 175 Phe Trp Glu Ser Val Phe Thr Gly Leu Thr His Ile Asp Ala His Phe             180 185 190 Leu Ser Gln Thr Lys Gln Ala Gly Asp Asn Phe Pro Tyr Leu Val Ala         195 200 205 Tyr Gln Ala Thr Val Cys Ala Arg Ser Gln Ala Pro Pro Pro Ser Trp     210 215 220 Asp Gln 225 <210> 14 <211> 678 <212> DNA <213> Hepatitis C virus fragment 7 <400> 14 ttcacagaca actcctcccc cccggcagta ccgcagacat tccaagtggc ccatctacac 60 gctcccaccg gcagtggcaa gagcaccaaa gtgccagctg cgtacgcagc ccaagggtac 120 gt; aaggcatatg gtaccgaccc caatgtcaga actggggtaa ggaccatcac cacgggcagc 240 cccatcacgt actcctctgg agggggttcc accattgaga cgacgaccgt gccccaagac 300 gcggtgtcgc gctcgcagcg gcgaggcagg actggtaggg gtagggcagg catctacagg 360 tttgtgactc cgggagaacg gccctcgggc atgttcgatt cctcggtcct gtgtgagtgt 420 tatgactcgg gctgtgcttg gtacgaactc acgcccgctg agacctcggt taggttgcgg 480 gcatacctaa atacaccagg gctgcccgtc tgtcaggacc atctggagtt ctgggagagt 540 gtcttcacag gcctcaccca catagatgcc cacttcttgt cccaaactaa gcaggcagga 600 gacaacttcc cctacctggt agcataccaa gctacagtgt gcgccaggtc tcaggcccca 660 cctccatcgt gggatcaa 678 <210> 15 <211> 303 <212> PRT <213> Hepatitis C virus fragment 8 <400> 15 Phe Thr Asp Ser Ser Pro Pro Ala Val Pro Gln Thr Phe Gln Val   1 5 10 15 Ala His Leu His Ala Pro Thr Gly Ser Gly Lys Ser Thr Lys Val Pro              20 25 30 Ala Ala Tyr Ala Ala Gln Gly Tyr Lys Val Leu Val Leu Asn Pro Ser          35 40 45 Val Ala Ala Thr Leu Gly Phe Gly Ala Tyr Met Ser Lys Ala Tyr Gly      50 55 60 Thr Asp Pro Asn Val Arg Thr Gly Val Arg Thr Ile Thr Thr Gly Ser  65 70 75 80 Pro Ile Thr Ser Ser Gly Gly Gly Ser Gly Gly Cys Ser Gly Gly                  85 90 95 Ala Tyr Asp Ile Ile Ile Cys Asp Glu Cys His Ser Thr Asp Ser Thr             100 105 110 Thr Ile Leu Gly Ile Gly Thr Val Leu Asp Gln Ala Glu Thr Ala Gly         115 120 125 Ala Arg Leu Val Val Leu Ala Thr Ala Thr Pro Pro Gly Ser Val Thr     130 135 140 Val Pro His Pro Asn Ile Glu Glu Val Gly Leu Ser Asn Thr Gly Glu 145 150 155 160 Ile Pro Ser Gly Gly Gly Ser Thr Ile Glu Thr Thr Thr Val Pro Gln                 165 170 175 Asp Ala Val Ser Arg Ser Gln Arg Arg Gly Arg Thr Gly Arg Gly Arg             180 185 190 Ala Gly Ile Tyr Arg Phe Val Thr Pro Gly Glu Arg Pro Ser Gly Met         195 200 205 Phe Asp Ser Ser Val Leu Cys Glu Cys Tyr Asp Ser Gly Cys Ala Trp     210 215 220 Tyr Glu Leu Thr Pro Ala Glu Thr Ser Val Arg Leu Arg Ala Tyr Leu 225 230 235 240 Asn Thr Pro Gly Leu Pro Val Cys Gln Asp His Leu Glu Phe Trp Glu                 245 250 255 Ser Val Phe Thr Gly Leu Thr His Ile Asp Ala His Phe Leu Ser Gln             260 265 270 Thr Lys Gln Ala Gly Asp Asn Phe Pro Tyr Leu Val Ala Tyr Gln Ala         275 280 285 Thr Val Cys Ala Arg Ser Gln Ala Pro Pro Ser Ser Trp Asp Gln     290 295 300 <210> 16 <211> 909 <212> DNA <213> Hepatitis C virus fragment 8 <400> 16 ttcacagaca actcctcccc cccggcagta ccgcagacat tccaagtggc ccatctacac 60 gctcccaccg gcagtggcaa gagcaccaaa gtgccagctg cgtacgcagc ccaagggtac 120 gt; aaggcatatg gtaccgaccc caatgtcaga actggggtaa ggaccatcac cacgggcagc 240 cccatcacgt actcctctgg agggggttcc ggcggttgct ctgggggcgc ctacgacatc 300 ataatatgcg atgagtgcca ctcaactgac tcgactacca tcttgggcat cggcacagtc 360 ctggaccaag cggagacggc tggagcgcgg ctcgtcgtgc tcgccaccgc tacacctccg 420 gggtcggtca ccgtgccaca cccaaatatc gaggaagtgg gcctgtccaa tactggggag 480 atcccctctg gagggggttc caccattgag acgacgaccg tgccccaaga cgcggtgtcg 540 cgctcgcagc ggcgaggcag gactggtagg ggtagggcag gcatctacag gtttgtgact 600 ccgggagaac ggccctcggg catgttcgat tcctcggtcc tgtgtgagtg ttatgactcg 660 ggctgtgctt ggtacgaact cacgcccgct gagacctcgg ttaggttgcg ggcataccta 720 aatacaccag ggctgcccgt ctgtcaggac catctggagt tctgggagag tgtcttcaca 780 ggcctcaccc acatagatgc ccacttcttg tcccaaacta agcaggcagg agacaacttc 840 ccctacctgg tagcatacca agctacagtg tgcgccaggt ctcaggcccc acctccatcg 900 tgggatcaa 909 <210> 17 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> NS3f1 forward primer <400> 17 ggatccacag acaactcctc cccc 24 <210> 18 <211> 33 <212> DNA <213> Artificial Sequence <220> <223> NS3f1 reverse primer <400> 18 ggaaccccct ccagatttgg ttatggggtg tgt 33 <210> 19 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> NS3f2 Forward primer <400> 19 ggatccttca cagacaactc ctcc 24 <210> 20 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> NS3f2 Reverse primer <400> 20 gtcgacttag gagtacgtga tggggct 27 <210> 21 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> NS3f3 Forward primer <400> 21 ggatccggcg gttgctctgg gggc 24 <210> 22 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> NS3f3 Reverse primer <400> 22 gtcgacttag gggatctccc cagtatt 27 <210> 23 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> NS3f4 Forward primer <400> 23 ggatccacca ttgagacgac gacc 24 <210> 24 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> NS3f4 Reverse primer <400> 24 gtcgacttat tgatcccacg atggagg 27 <210> 25 <211> 5 <212> PRT <213> Artificial Sequence <220> <223> peptide linker <400> 25 Ser Gly Gly Gly Ser   1 5 <210> 26 <211> 15 <212> DNA <213> Artificial Sequence <220> <223> linker <400> 26 tctggagggg gttcc 15 <210> 27 <211> 15 <212> DNA <213> Artificial Sequence <220> <223> linker <400> 27 agcggggggg ggagc 15 <210> 28 <211> 15 <212> DNA <213> Artificial Sequence <220> <223> linker <400> 28 agcggtggtg gcagt 15 <210> 29 <211> 15 <212> DNA <213> Artificial Sequence <220> <223> linker <400> 29 tccgggggtg gttca 15 <210> 30 <211> 15 <212> DNA <213> Artificial Sequence <220> <223> linker <400> 30 tcaggtggcg ggtcg 15

Claims (12)

A separate fusion polypeptide comprising three or more hydrophilic fragments which are contiguous amino acid sequences selected from the amino acid sequence of hepatitis C virus non-structural protein 3 (HCV NS3)
Wherein said hydrophilic fragment is comprised of the amino acid sequence of SEQ ID NO: 3, SEQ ID NO: 5, or SEQ ID NO: 7. 2. The fusion polypeptide of claim 1, wherein said HCV NS3 is a C-terminal domain comprising the helicase activity of HCV NS3 consisting of the amino acid sequence of SEQ ID NO: 1. delete 2. The fusion polypeptide of claim 1, wherein each of the three or more hydrophilic fragments is linked by a peptide linker. 5. The fusion polypeptide of claim 4, wherein the peptide linker is a polypeptide consisting of 2 to 100 amino acids. 2. The fusion polypeptide of claim 1, wherein the fusion polypeptide comprises the amino acid sequence of SEQ ID NO: 3. The fusion polypeptide of claim 1, wherein the fusion polypeptide binds to an antibody in a subject having hepatitis C virus infection. An isolated polynucleotide encoding the fusion polypeptide of claim 1. A composition for diagnosing hepatitis C virus infection comprising the fusion polypeptide of any one of claims 1, 2, and 4 to 7. A kit for diagnosing hepatitis C virus infection comprising the fusion polypeptide of any one of claims 1, 2, and 4 to 7. Contacting a sample isolated from an individual with the fusion polypeptide of any one of claims 1, 2, and 4 to 7; And
And detecting the presence of the complex of the fusion polypeptide and the hepatitis C virus antibody in the sample to detect the hepatitis C virus antibody. 12. The method according to claim 11, wherein the sample is blood, whole blood, serum or plasma of an individual.