KR100605322B1 - Antigenic Epitope for Diagnosis of Herpatitis C Virus Antibody - Google Patents

Abstract

The present invention relates to a highly sensitive core antigenic epitope useful for the determination of hepatitis C virus (HCV) antibodies, and more specifically, Pseudomonas syringae-derived ice nucleus protein (expressed on Pseudomonas syringae) E. coli cell surface expression technology of foreign protein using ice nucleation protein (INP) is used to express the major antigenic epitope of HCV core protein on E. coli cell surface and HCV anti-core A technique for detecting antibodies rapidly and economically.

HCV, ice core active protein, cell surface expression, anti-core antibody, epitope

Description

Antigenic Epitope for Diagnosis of Herpatitis C Virus Antibody for Use in Diagnosing Hepatitis Virus Anti-Coa Protein Antibodies             

1 is a manufacturing method and structure of an ice nucleus active protein expression vector capable of expressing a protein on the cell surface of E. coli used in the present invention.

2 is a schematic diagram of a recombinant surface expression vector (pT7INC-core) in which a gene encoding HCV core protein used in the present invention is fused to an INC (recombinant protein combining N- and C-terminal INP) genes 3 '.

Figure 3 is a schematic diagram of the nine antigenic epitopes of HCV core structural proteins used to prepare HCV core antigenic epitope surface expression vector in the present invention.

Figure 4 shows the results confirming that the INC-c22p fusion of the INC protein or the existing HCV core antigenic epitope to the carboxyl terminal of the INC using the surface expression vector is expressed on the cell surface of E. coli. Figure 4 (A) through immunofluorescence microscopy, Figure 4 (B) and Figure 4 (C) is Western blotting analysis using the serum of INP antibody and HCV infected patients, respectively, INC or INC-c22p The surface expression was confirmed.

5 shows the results of Western blotting confirming the expression of antibodies and the recognition of major antigenic epitope fragments of HCV core structural proteins. Figure 5 (A) is the result of Western-broken with an antibody against the ice nucleus active protein after electrophoresing the entire recombinant E. coli cell protein expressing the fusion protein in SDS-polyacrylamide gel. FIG. 5 (B) shows a core antigen expressed as a fusion protein by Western blotting using the sera of HCV chronic hepatitis patients having a specific antibody to the core antigen after electrophoresis of the protein as shown in FIG. 5 (A). This is the result of analyzing the specific reaction between sex epitopes and antibodies.

FIG. 6 shows an enzymatic immunosorbent assay (ELISA) using E. coli cells expressing antigens on the cell surface to determine the specific binding level between recombinant HCV core protein major antigenic epitopes and antibodies in serum of HCV positive patients. The result is.

FIG. 7 shows a method for fluorescence-activated cell selection (FACS: fluorescence activated cell) in which the core antigen is expressed on the surface of E. coli cells using the sera of HCV infected patients in which the specific antibody to the core antigen is present to confirm the surface expression of the superior antigen. measured by sorter).

FIG. 8 shows a comparison of the core HCV core antigenic epitopes (c22p, C2, and C3) with the ability to detect core specific antibodies present in the serum of HCV infected patients by ELISA analysis using 10 HCV infected patient sera. This is a result confirming the superiority to novel HCV core antigenic epitopes C2 and C3 that can detect antibodies with higher sensitivity than c22p, a sex epitope.

Field of invention

The present invention relates to a highly sensitive core antigenic epitope useful for the determination of Hepatitis C virus (HCV) antibodies. More specifically, by using a technique for expressing a foreign protein on the surface of E. coli cells, the important core antigenic epitopes of HCV can be expressed on the surface of E. coli and the antibody can be detected. The present invention relates to a method for quickly and economically identifying antigenic epitopes that respond more sensitively than the core antigenic epitopes used.

Background of the Invention

Hepatitis virus, one of the various pathogen viruses on earth, is known to be infected by blood transfusion, injection or childbirth. Many cases of viral hepatitis due to blood transfusion have been reported to date, and such viral hepatitis is generally classified into types A, B and non-A, and non-B. Conventionally, hepatitis B virus (HBV) is known as a cause of viral hepatitis when transfused. Recently, test reagents for detecting the presence of such HBV have been developed, and since there is no HBV infection due to blood transfusion, the presence of HBV can be easily confirmed before transfusion. On the other hand, the presence of hepatitis A (non-A, non-B) due to hepatitis A virus (HAV) or hepatitis B virus (HBV), which is clearly a virus and is thought to cause infection. The presence of non-A, non-B hepatitis viruses has been clarified by the literature in 1989. Choo, QL et al., Science, 244: 359-362 (1989); Kuo, G. et al., Science, 244: 362-364 (1989). Infection with HCV is reported to have a high incidence of chronic hepatitis and may lead to cirrhosis and hepatocellular carcinoma as the duration of infection continues. Choo, QL et al., Science, 244: 359-362 (1989); Kuo, G. et al., Science, 244: 362-364 (1989); Saito, L. et al., Proc. Natl. Acad. Sci. USA, 87: 6547-6549 (1990).

HCV is a single stranded (+) RNA virus with a gene of about 9.5 kb in size and belongs to the Flaviviridae family of genes associated with flavivirus or pestivirus. When a cell is infected by HCV RNA, it is translated into HCV RNA to form a single large polypeptide consisting of 3011 amino acids, which is a core protein (18-22 kDa) by a host cell or virus-derived protease. ) And glycosylated envelope proteins E1 (31-35 kDa) and E2 (58-74 kDa) structural proteins and five nonstructural proteins (NS2, NS3, NS4A, NS4B, NS5A, NS5B). Fournillier, JA et al., J. Gen. Virol., 77: 1055-1064 (1996); Grakoui, A. et al., J. Virol., 67: 1385-1395 (1993); Lanford, R. E. et al., Virology, 197: 225-235 (1993); Matsura, Y. et al., Virology, 205: 141-150 (1991).

At present, the infection of HCV is mainly caused by the blood, so the path of infection can be blocked by detecting and excluding infectious agents. Currently, methods for detecting infectious agents are mainly used to detect HCV antibodies in a sample and to detect HCV gene RNA by using an immunoassay using specific binding of HCV antibodies to HCV polypeptides. In the antigen detection method, core antigen is widely used for detection of HCV, and core protein is a nucleocapsid that surrounds HCV RNA, showing high antigenicity, and amino acid sequence difference among several genotypes of HCV. Are reported to be the least (Houghton, M. et al., Hepatology, 14: 381-388 (1991)). In addition, since the mutation rate is lower than that of other antigens E1 and E2, detecting core antigenic epitopes is very essential and effective for detecting HCV antibodies. As the antigen used for detecting the core antibody, a peptide containing a HCV core antigenic epitope (c22p: amino acids 10 to 53 in the core protein of SEQ ID NO: 1) or a polypeptide is used. However, in many cases, the sensitivity and precision of antibody detection methods for detecting HCV infection are still inaccurate, and thus a more accurate and effective method for detecting HCV infection is required. In addition, the development of a technology that can replace the method of finding an antigenic epitope using a time-consuming random peptide expression phage library, which requires a phage propagation step, or a peptide screening method of chemically synthesizing a large number of expensive antigenic epitopes. This is necessary.

Accordingly, the present inventors developed and used a microbial cell surface expression method to develop an antigenic epitope capable of detecting antibodies more sensitive than the conventional c22p and HCV core antigenic epitopes used for detecting antibodies against HCV. The microbial surface expression system using the ice nucleus active protein used for this purpose is a technology for stably expressing foreign proteins on the surface using a surface anchoring motif, and it is possible to express a large amount of proteins that are difficult to produce. These proteins can be used for antibody detection using the properties of E. coli cell surface.

Pseudomonas Schringe-derived nucleus-activated protein has a sequence of 8, 16 or 48 amino acids in the center of the protein structure, which provides a framework for supercooled water molecules to arrange ice particles well, At the N- and C-terminus, there is a secretion signal and a target signal, allowing proteins to cross the cell membrane. Specifically, the nucleus nucleus activating protein is composed of a total of 1,200 amino acids. The repeat sequence between the N-terminus and the C-terminus is related to the ice nucleus activity, and its length can be controlled, and the N-terminus is simply the outer membrane. It has been reported that the C-terminus secretes and targets proteins to the outer membrane of membranes, which has a function of attaching to them [Green, RL, et al., Mol. Gen. Genet., 215: 165-172 (1988). The ice-nucleated active protein can be usefully used as a surface expression matrix capable of expressing foreign proteins on the cell surface due to the above characteristics.

Therefore, the present inventors expressed the surface expression using N-terminal (15% of total protein) and C-terminal (4% of total protein) of Pseudomonas syringae (KCTC 1832 BP) -derived ice core active proteins. Vectors were constructed and used to express major core antigenic epitopes of HCV on the surface of Escherichia coli [E. coli BL21 (DE3)], which were then more efficient than synthetic peptides (c22p) used in conventional diagnostics for HCV core antibody detection. The present invention was completed by discovering highly sensitive antigenic epitopes that can be diagnosed and confirming their antibody detection excellence.

An object of the present invention is to provide a highly sensitive HCV core antigenic epitope and HCV detection kit containing the epitope that can be used in the HCV antibody measurement method for detecting HCV infection.

Another object of the present invention is to provide an oligonucleotide encoding the epitope and a cell surface expression vector containing the gene of the oligonucleotide and secretory induction protein.

Another object of the present invention is to provide a surface expression vector (pT7INC-core, pT7INC-c22p, pT7INC-C1 to pT7INC-C8) capable of producing HCV core antigen protein and a microorganism transformed with the expression vector.                         

Another object of the present invention is to provide a method for screening antigenic epitopes with high antibody recognition activity.

In the present invention, C1 is a peptide consisting of 38th to 53rd amino acids of HCV core protein having the amino acid sequence of SEQ ID NO: 1, C2 is 26th to 53rd amino acid, C3 is 1st to 38th amino acid, and C4 is A peptide consisting of amino acids 101 to 121, C5 is 38th to 75th amino acid, C6 is 1st to 26th amino acid, C7 is 1st to 75th amino acid, and C8 is 38th to 121th amino acid. to be.

In the present invention, cell surface expression technology was used to develop a core antigenic epitope that is more effective than a conventional core antigenic epitope used for HCV core antibody measurement in the conventional HCV detection method. Cell surface expression technology is a method to connect the appropriate extracellular membrane protein and foreign protein at the gene level to induce the fusion protein biosynthesis, and then stably pass through the intracellular membrane to remain attached to the extracellular membrane. This surface expression method has an advantage in that it is easy to measure the activity of the protein and the expression level is uniformly controlled by the anchor protein fused to the protein of interest and is difficult to express the protein.

 In order to measure the specific antibody binding capacity of the core antigen, a cell surface expression vector into which a major core antigenic epitope gene was inserted was prepared. Encoding the N-terminal and C-terminus using the existing plasmid vector pGINP21 (Accession No .: KTCT No. 8608P) cloned with a Pseudomonas Schringe-derived ice core active protein gene to obtain a surface expression vector using an ice core active protein The gene was amplified by a polymerase chain reaction (PCR), and inserted into a pET22b (+) vector where transcription was initiated by a T7 promoter to prepare a surface expression vector. It is a vector produced to induce expression by controlling the expression by lacI repressor and adding isopropyl-beta-di-thiogalactosidase (isoTG-(-D-thiogalactosidase, IPTG) to the medium.

In the present invention, amplification of genes corresponding to core antigens is performed by PCR from HCV cDNA derived from RNA extracted from serum of Korean HCV-positive patients, and these genes are inserted into the surface expression vector pT7INC by adjusting the translational codons to the ice core active protein coding genes. PT7INC-core plasmid was produced by this. In addition, the pT7INC-core was used as a template to insert the c22p coding gene, which is the main antigenic epitope of the HCV core, and the epitopes of C1 to C8 according to the present invention into the surface expression vector pT7INC, thereby inserting pT7INC-c22p and pT7INC-C1 to pT7INC. -C8 was produced.

In order to confirm that the core antigen protein and fragments thereof are properly synthesized in the cell and stably attached to the cell surface through the inner membrane, the E. coli BL21 (DE3) is transformed with the expression vector, and then cultured. Protein synthesis was induced to surface express major antigenic epitopes in HCV core antigens.

Immunofluorescence microscopy and Western blotting analysis with serum of HCV infectious agents in which antibodies to core antigens were present confirmed that HCV core antigens and antigenic epitopes were stably expressed on the cell surface.

Whether or not these major antigenic epitopes were expressed on the cell surface was confirmed by fluorescence-activated cell screening (FACScan). As a result, core antigenic epitope fragments were expressed on the cell surface, and the fusion protein was expressed on the HCV core on the E. coli cell surface. It was confirmed that the specific reaction with the antibody against.

As a result, the present invention is characterized by providing an antigenic epitope for detecting HCV selected from the group consisting of C2, C3, C6 and C7. The present invention also provides fusion proteins in which the oligonucleotides encoding the epitopes, the same two or more epitopes of the epitopes are fused, or two or more different epitopes are fused.

Fusion proteins can be produced by conventional recombinant DNA techniques. That is, a recombinant expression vector containing a gene of the protein to be fused with an oligonucleotide encoding the epitope is prepared, and then the recombinant expression vector is introduced into a suitable host cell to express the fusion protein by conventional techniques. Suitable host cells for the expression of the fusion protein are Escherichia coli, bacteria such as Bacillus subtilis and Bacillus brevis, actinomycetes such as Streptomyces lividans, yeasts such as Streptomyces cerevisiae, Aspergillus oriza and Aspergillus. Fungi such as snidulans and Aspergillus niger, animal cells such as COS-7, CHO, Vero, mouse L cells, insect cells, plant cells and the like can be used. The host cell used in the present invention is preferably E. coli, more preferably E. coli BL21 (DE3) can be used. The protein to be fused is not particularly limited, but it is preferable to use a secretion-inducing protein capable of inducing expression on the cell surface.

The present invention also includes a primer pair selected from the group consisting of SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, and SEQ ID NO: 17 and the epitope or the fusion protein useful for synthesizing the oligonucleotide. To provide a kit and composition for detecting HCV. Kit for detecting HCV comprising the epitope or fusion protein of the present invention is a conventional method, enzyme immunosorbent assay (ELISA), fluorescence-activated cell screening method (FACS), live cell panning (screening) after screening, green fluorescent protein ( A substance capable of measuring the degree of binding, such as a green fluorescence protein), can be prepared by various methods, such as a method of expressing HCV antibody detection level by the expression of fluorescence in the cell together.

The present invention also provides a cell surface expression vector containing the gene of the oligonucleotide and secretion induction protein. The present invention is characterized in that it provides a method for expressing the microorganism transformed with the expression vector and the antigenic epitope characterized in culturing the microorganism on the cell surface. The microorganism used at this time is preferably E. coli which can recognize the promoter of the vector, more preferably E. coli BL21 (DE3) can be used.

The present invention also provides a microorganism characterized in that the antigenic epitope is expressed on the surface, and a kit and a composition for detecting HCV containing the microorganism. The secretion inducing protein may be an ice nucleus active protein or part thereof. In addition, the ice nucleus active protein may be derived from Pseudomonas Shuringe, and a portion of the ice nucleus active protein may be the N end of the cell attachment site and a site C end having secretion and target function to the extracellular membrane. The N- and C-terminal DNA of the ice core active protein may be fragments cut by HindIII and BamHI.

Preferred cell surface expression vectors for use in the present invention are the genes described in FIG. 1 prepared by inserting the N- and C-terminal genes of the ice core active protein cut with HindIII and BamHI into the base vector pET22b (+) cut with HindIII and BamHI. It is a surface expression vector pT7INC with a map. The vector may contain a DNA encoding an antigenic protein or part thereof of a pathogenic virus or microorganism, and the DNA encoding the antigenic protein or part thereof may be an oligonucleotide encoding an epitope of the present invention or a gene of HCV core protein. Or DNA encoding c22p. The expression vector is composed of pT7INC-core, pT7INC-c22p, pT7INC-C1, pT7INC-C2, pT7INC-C3, pT7INC-C4, pT7INC-C5, pT7INC-C6, pT7INC-C7 and pT7INC-C8 have.

In another aspect, the present invention is characterized by providing a method for expressing the microorganism transformed with the expression vector and the antigenic epitope characterized in culturing the microorganism on the cell surface. The microorganism used at this time is preferably E. coli which can recognize the promoter of the vector, more preferably E. coli BL21 (DE3) can be used.

In addition, the present invention comprises the steps of (a) preparing a library of DNA encoding the antigenic protein or part of the pathogenic virus or microorganism; (b) inserting the library into a cell surface expression vector containing DNA encoding a cell adhesion site (N terminus) of the nucleus nucleus-active protein and a site (C terminus) secreted to the outer membrane and a target function (terminal C) Producing a; (c) transforming each microorganism with the vector library; (d) culturing the transformed microorganism to express the antigenic protein or part thereof on the cell surface; And (e) detecting and selecting an antigenic epitope that specifically binds to the serum of a patient infected with the pathogenic virus or microorganism.

Here, the DNA encoding the antigenic protein or part of the pathogenic virus or microorganism may be a gene of HCV core protein or a fragment thereof, and secrete and target function of the ice nucleus active protein to the cell attachment site (N-terminal) and the outer membrane of the cell. The cell surface expression vector containing the DNA encoding the region (terminal C) having it may be pT7INC.

In this method, a DNA library refers to all DNAs modified using an antigenic protein gene. Such a method of preparing a DNA library may be prepared by analyzing the structure of the antigenic protein and amplifying the parts predicting that the antigenic epitope may exist, or by generating random mutations or randomly cutting the antigenic DNA. . At this time, as a method of generating a random mutation, DNA shuffling, error-prone PCR method, and the like can be used. In addition, a part of the antigen may be selected to bind the same sites to each other to prepare a multimer.

One of the reasons for the recent increase in the number of HCV-infected patients worldwide is that there are no well-defined infectious agents, that is, highly sensitive diagnostic methods, to prevent the spread of these infectious agents due to blood transfusion. In the absence of a prophylactic vaccine and an effective therapeutic agent, the core antigenic epitope found in the present invention, which is more sensitive than the synthetic core antigen peptide (c22p) used in the core antibody measurement method, can be effectively used for diagnostic reagent development and live vaccine development. It is expected to be.

Hereinafter, the present invention will be described in more detail with reference to Examples. These examples are only for illustrating the present invention, and the scope of the present invention will not be construed as being limited by these examples.

Example 1 Preparation of Surface Expression Vector pT7INC

A novel expression vector plasmid using a T7 promoter was prepared to produce an E. coli high expression vector using an INC (recombinant protein combining the N- and C-terminus of INP) recombinant anchor motif consisting of the N- and C-terminus of the ice core active protein. It was. The INC protein gene was obtained from the existing ice core active protein expression vector plasmid pANC3 (KCTC 0326 BP). First, pET-22b (+) (Novazen, Germany) and pANC3 plasmids containing the T7 promoter were digested with NdeI and HindIII, respectively, and then blunt-ended using Klenow fragment. Subsequently, both DNAs were cut with BamHI to obtain a vector region and an INC gene region containing a T7 promoter, a replication start site, an antibiotic resistance gene, and the like, and the two DNA fragments were combined to obtain a recombinant vector of about 6.4 kbp, pT7INC. It was produced (Fig. 1).

Example 2 Preparation of Recombinant Fusion Protein Surface Expression Vectors pT7INC-core, pT7INC-c22p, pT7INC-C1-8

In order to secure the Korean HCV gene, cDNA was synthesized using random hexamer and reverse transcriptase as a template of total RNA including HCV RNA gene isolated from infected patient serum. After mixing RNA and random hexamer, it was heated at 70 ° C. for 5 minutes and cooled rapidly on ice. The mixed solution was incubated at 37 ° C. for 60 minutes with reverse transcriptase buffer, 1 mM dNTP, and 20 U reverse transcriptase, and inactivated at 95 ° C. for 5 minutes. Total cDNA of the core protein (SEQ ID NO: 1) of HCV was amplified under the following conditions.

HCV core protein amino acid sequence: (SEQ ID NO: 1):

MSTNPKPQRKTKRNTNRRPQDVKFPGGGQIVGGVYLLPRRGPRLGVRATRKASERSQPRGRRQPIPKARRPEGRAWAQPGYPWPLYGNEGLGWAGWLLSPRGSRPSWGPTDPRRRSRNLGKVIDTLTCGFADLMGYIPLVGAPLGGAARALAVFSASGGLS

PCR was performed using oligonucleotides having the nucleotide sequences of SEQ ID NO: 2 and SEQ ID NO: 3 at 54 ° C for 1 minute 30 seconds, 92 ° C for 1 minute 30 seconds, 92 ° C for 1 minute 30 seconds, and 72 ° C for 2 minutes 30 seconds. 40 PCR reactions were performed. The amplified DNA was digested with restriction enzymes BamHI and EcoRⅠ and then inserted into the pT7INC vector containing the T7 promoter digested with the same enzymes to produce a 6.9 kbp expression vector having the entire core antigen gene at the end of the INC gene 3 '. It was named surface expression vector pT7INC-core (Fig. 2).

SEQ ID NO: 5'-GATTGGGGGCGACACTCCACC-3 '

SEQ ID NO: 5'-GGAATTCCTTAAGCGGAGGCTGGGATGGTC-3 '

Figure 3 is a schematic diagram of the major antigenic epitope fragments of HCV core structural protein used to prepare a core antigenic epitope surface expression vector in the present invention. The antigenic epitopes C1 to C8 are further constructed around the hydrophilic region of the core antigen. Black areas indicate hydrophilic sites in HCV core protein.

pT7INC-C1 was prepared by PCR using the expression plasmid (pT7INC-core) as a template and oligonucleotides of SEQ ID NO: 4 and SEQ ID NO: 5 as primers (1 minute at 94 ° C, 1 minute at 55 ° C, 1 minute at 72 ° C). Conditions of the 45 bp core protein fragment were digested with restriction enzymes BamHI and EcoRⅠ, followed by N repeats of T7 promoter and ice nucleus activating protein. It is a surface expression vector of about 6.45kbp obtained by inserting into a pT7INC expression vector composed of genes encoding the terminal and C-terminal sites. pT7INC-C2 was prepared in the same manner after using p7INC-core as a template, PCR of the oligonucleotides of SEQ ID NO: 6 and SEQ ID NO: 7 with a primer. Similarly, for pT7INC-C3, SEQ ID NO: 8 and SEQ ID NO: 9 are primers; for pT7INC-C4, SEQ ID NO: 10 and SEQ ID NO: 11 are primers; for pT7INC-C5, SEQ ID NO: 12 and 13 are primers; pT7INC-C6 In the case of SEQ ID NO: 14 and SEQ ID NO: 15 as a primer, and in the case of pT7INC-C7, SEQ ID NO: 16 and SEQ ID NO: 17 as a primer, and in the case of pT7INC-C8, SEQ ID NO: 18 and SEQ ID NO: 19 as a primer to prepare a vector . In the case of the previously used core antigen c22p was cloned by the same method using SEQ ID NO: 20 and SEQ ID NO: 21 as a primer and named pT7INC-c22p (Fig. 3).

SEQ ID NO: 5'-CGGGATCCGCGCAGGGGCCCCAGGTTGGGTGTG-3 '

SEQ ID NO: 5'-GGAATTCCTTAGGAAGTCTTCCTAGTCGCGC-3 '

SEQ ID NO: 5'-CGGGATCCGGGCGGTGGTCAGATCGTTGGTGGA-3 '

SEQ ID NO: 5'-GGAATTCCTTAGGAAGTCTTCCTAGTCGCGC-3 '

SEQ ID NO: 8'-GATTGGGGGCGACACTCCACC-3 '

SEQ ID NO: 5'-GGAATTCTTACGGCAACAGGTAAACTCCACCAAC-3 '

SEQ ID NO: 10'-CGGGATCCCCGTGGCTCCCGGCCTAGTTGGGGC-3 '

SEQ ID NO: 11'5'-GGAATTCCTTAACCCAAGTTACGCGACCTAC-3 '

SEQ ID NO: 12'-CGGGATCCGCGCAGGGGCCCCAGGTTGGGTGTG-3 '

SEQ ID NO: 13'5'-GGAATTCTTACCAGGCCCTACCCTCGGGCTGGCG-3 '

SEQ ID NO: 14'-GATTGGGGGCGACACTCCACC-3 '

SEQ ID NO: 15'-GGAATTCTTACGGGAACTTGAACGTCCTGTGGGCG-3 '

SEQ ID NO: 16: 5'-GATTGGGGGCGACA CTCCACC-3 '

SEQ ID NO: 17'-GGAATTCTTACCAGGCCCTACCCTCGGGCTGGCG-3 '

SEQ ID NO: 18'-CGGGATCCGCGCAGGGGCCCCAGGTTGGGTGTG-3 '

SEQ ID NO: 19'5'-GGAATTCCTTAACCCAAGTTACGCGACCTAC-3 '

SEQ ID NO: 20'-CGGGATCCCAAAACCAAACGTAACACCAA-3 '

SEQ ID NO: 21'5'-GGAATTCCTTAGGAAGTCTTCCTAGTCGCGC-3 '

Example 3 Expression of Major Antigenic Epitopes in HGV Core Proteins

In order to investigate whether core antigens are expressed on the surface of Escherichia coli cells in E. coli transformed with the surface expression vectors pT7INC-core, pT7INC-c22p, and pT7INC-C1 to pT7INC-C8, the antigens are expressed on the surface of E. coli cells to be reactive with antibodies. Enzyme genes were identified using E. coli BL21 (DE3) on chromosomal DNA. Each recombinant plasmid DNA was introduced into BL21 (DE3) strain by heat shock at 42 ° C. for 90 seconds. Recombinant BL21 (DE3) cells with recombinant plasmid DNA were introduced into 5 ml of LB medium (5 g / l yeast extract, 10 g / l tryptone, 5 g / l salt, pH 7.0) with 0.1 mg / ml of antibiotic ampicillin. ) Was incubated for 12 hours at 37 ℃, 200 rpm shaking culture conditions. The culture solution was diluted 1: 100 in 5 ml LB medium supplemented with 0.1 mg / ml of antibiotic ampicillin and incubated for 2 to 3 hours at 37 ° C and 200 rpm. T7 RNA when strain growth was 0.4 to 0.5 at 600 nm. To induce polymerase expression, IPTG was added at a concentration of 1 mM to induce expression of the protein at 25 ° C. for 6 hours.

Example 4 Confirmation of Cell Surface Expression of INC Fusion Protein Using Immunofluorescence Microscopy

Recombinant cells expressing INC or INC-c22p were cultured and washed with PBS buffer, and then suspended in PBS buffer containing 3% BSA with an absorbance of 0.5 at 600 nm. The serum of HCV infected patients was diluted 1: 1000 and reacted at 4 ° C. for 12 hours, and then washed 5 times with PBS. Next, FITC bound anti-human IgG (immunoglobulin) was diluted 1: 3000 and suspended in PBS buffer containing 3% BSA and reacted at 37 ° C for 2 hours. After washing five times with PBS buffer again, the degree of fluorescence of the cells was measured with a confocal laser microscope. From the above experiments, it was confirmed that c22p, a representative HCV core antigenic epitope, was stably expressed on the cell surface (FIG. 4).

4 shows the results of confirming the expression of recombinant protein INC or recombinant antigen protein INC-c22p on the surface of E. coli cells using an expression vector. 4 (A) shows whether INC or INC-c22p is expressed on the surface of E. coli as a single antibody that recognizes (His) 6 at the carboxy terminus, and INC-c22p is reacted with serum of HCV infected patients. The FITC-coupled anti-mouse and anti-human Ig, respectively, treated with an immunofluorescence microscope to measure the degree of fluorescence. Figure 4 (B) is the result of confirming the expression of the fusion protein by Western blotting using the antibody against the ice core active protein after electrophoresis of the recombinant E. coli protein body on the SDS-polyacrylamide gel. FIG. 4 (C) shows the results obtained by Western blotting analysis of electrophoresis of proteins in the same manner as FIG. 4 (B) and the anti-core antibody in serum of HCV infected patients with specificity for c22p. 4 (B) and 4 (C), 1 is a result of analysis of Western protein by electrophoresis of the protein fraction of the strain into which the pT7INC expression vector is inserted and 2 into which the pT7INC-c22p is inserted.

Example 5: Confirmation of expression of fusion protein through western brotting and discovery of core antigenic epitopes that specifically react with serum of HCV infected patients

The expression of core antigen fused to an INC protein having N- and C-terminus of ice nucleating protein was confirmed by SDS-polyacrylamide gel electrophoresis and Western blotting using an antibody against core antigen. E. coli cells induced protein expression were centrifuged and dissolved in protein assay solution (50 mM Tris-Cl, 100 mM dithiothreitol, 2% SDS, 0.1% bromophenol blue, 10% glycerol) After heat treatment at 100 ° C. for 10 minutes, electrophoresis was performed. Next, the protein in the gel was broth onto a nitrocellulose membrane and treated for 1 hour in a blocking solution in which 2% BSA (bovine serum albumin) was added to TBST buffer solution (20 mM Tris-Cl, 150 mM NaCl, 0.05% Tween20). Non-specific binding was prevented, and the serum of anti-INP rabbit antibody or HCV infected patients was diluted in TBST solution at a ratio of 1: 1000 and reacted at room temperature for 1 hour. The nitrocellulose membranes were then washed three times with TBST buffer three times for 10 minutes, followed by 1 hour at room temperature with anti-rabbit Ig or anti-human Ig conjugated with alkaline phosphatase (ALP) diluted by TBST solution at a ratio of 1: 5000. Reacted for a while. Then, the membrane was washed three times with TBST buffer and then NBT / BCIP (Sigma, USA) was used to detect the antibody-reactive protein.

As shown in FIG. 5 from the above experimental results, all recombinant strains were recognized by the anti-INP antibody, and it was confirmed that all the fusion proteins were expressed on the cell surface with the expected size using the INC protein as the fusion parent.

In addition, Western blotting analysis was performed on serum derived from HCV infected patients in which the antibody against the ice core active protein and the antibody to the core antigen were present to perform fusion proteins (pT7INC-C1 to pT7INC-C8: about 41-63 kDa). It was confirmed that the specific reaction with the antibody to the protein and antibody derived from HCV infected patients (Fig. 5).

FIG. 5 (A) shows the results of Western blotting analysis with antibodies to the ice core active protein after electrophoresis of each E. coli cell protein fraction expressed on the cell surface using the ice core active protein gene.

FIG. 5 (B) shows a core antigenic epitope that specifically binds to the serum of a patient by performing a Western blotting analysis using the serum of HCV chronic hepatitis patients after electrophoresis of the protein as shown in FIG. 5 (A). One result is shown.

In FIG. 5 (A) and FIG. 5 (B), INC is a fraction obtained by Western-broken after electrophoresis of the E. coli cell protein fraction on SDS-polyacrylamide gel by inducing the expression of the transformant into which the pT7INC expression vector is inserted. PT7INC-C1 for C1, pT7INC-C2 for C2, pT7INC-C3 for C3, pT7INC-C4 for C4, pT7INC-C5 for C5, pT7INC-C6, C7 for In the case of pT7INC-C7 and in the case of C8, E. coli transformed with pT7INC-C8, respectively, to induce protein expression, and then the cell protein fractions of these E. coli were electrophoresed on SDS-polyacrylamide gel. Western broth analysis results.

As a result, it was found that the C2, C3, C6 and C7 sites in the anti-core antibody present in the sera of HCV infected patients include antibody recognition sites that specifically bind to the anti-core antibody as compared to other peptide antigenic epitopes. Could.

Example 6: ELISA using Escherichia coli expressing antigenic epitopes on the cell surface: Analysis of cell surface expressed fusion protein with anti-core antibody

Recombinant Escherichia coli induced fusion protein expression was suspended in PBS buffer solution at 600 nm so that the absorbance was 1.0, and 200 µl of the suspension per well was added to a 96-well microtiter plate (Nunc) and 12 at 37 ° C. The cells were allowed to solidify on a microplate by standing for time. After removing the PBS buffer with excess unsolidified cells, 200 μl of PBS buffer containing 2% BSA was added to each well, and reacted at 37 ° C. for 1 hour to prevent nonspecific binding. The buffer was removed and the serum of HCV infected patients was diluted 1: 1000 in PBS buffer, and 180 µl was added per well, followed by reaction at 37 ° C for 2 hours. The non-binding material was then removed, and each well was washed three times with PBS buffer, followed by the addition of 180 μl per well of PBS solution in which ALP-bound anti-human Ig was diluted in a 1: 5000 ratio at 37 ° C. The reaction was carried out for 2 hours. Subsequently, the unbound material was removed, and each well was washed three times with PBS buffer solution, and then 160 µl of NPP (Sigma) was added to induce a color reaction, and the color development was measured at an absorbance of 405 nm.

Figure 6 shows the results of the enzyme immunosorbent assay (ELISA) to determine the degree of specific binding between recombinant HCV core protein major antigenic epitopes and anti-core antibody in the serum of HCV positive patients.

Enzyme immunosorbent assay (ELISA) was used to determine the cell surface expression of core antigen and specificity of HCV anti-core antibody. The major antigenic epitopes were expressed on the surface of E. coli live cells and anti-core in HCV serum. It was confirmed that the antibodies specifically recognized them.

This suggests that C2 and C3 core antigenic epitopes have an excellent antibody recognition site that specifically binds to anti-core antibodies present in the sera of HCV infected patients compared to core peptide antigenic epitopes bound to other INC proteins. Could be (FIG. 6).

Example 7: Confirmation of cell surface expression of fusion protein by fluorescence-activated cell screening method (FACS)

These core proteins were used with recombinant E. coli BL21 (DE3) with recombinant plasmid pT7INC-core and pT7INC-C2 and pT7INC-C3, and wild line BL21 (DE3) without recombinant plasmid, respectively, as a control, 1 × 10 ⁸ , respectively. And whether the epitope is expressed on the cell surface. As shown in Example 3, E. coli, which induced protein expression, was harvested at the same concentration of 1 × 10 ⁸ cells, and suspended therein in PBS buffer diluted 1: 1000 with infected patient's serum. And then washed three times with PBS buffer. Next, the reaction was performed at 4 ° C. for 20 minutes using PBS buffer containing 1: 5000 anti-human Ig conjugated with FITC. Again washed three times with PBS buffer solution and analyzed for the degree of fluorescence.

7 is a result of measuring the recombinant fusion protein surface-expressed in Escherichia coli by fluorescent-active cell screening method using the serum of HCV infected patients with a specific antibody to the core antigen. The X axis of each graph represents fluorescence and the Y axis represents cell number.

Figure 7 (A) is the fluorescence degree of wild strain BL21 (DE3).

Figure 7 (B) is the degree of fluorescence of the recombinant strain introduced pT7INC-C2.

Figure 7 (C) is the degree of fluorescence of the recombinant strain introduced pT7INC-C3.

Figure 7 (D) is the degree of fluorescence of the recombinant strain in which pT7INC-core is introduced.

In comparison with the non-transfected Escherichia coli, the major superior core antigenic epitopes C2 and C3 and its precursor core protein, which were identified in the present invention, were transformed into E. coli in the E. coli transformed by various surface expression vectors. It was confirmed that the E. coli is expressed on the surface (Fig. 7).

Example 8 Analysis of Reactivity of Cell Surface-Expressed Fusion Proteins with Anti-Coa Antibodies by ELISA

Escherichia coli transformants expressing the fusion INC-c22p, INC-C2, INC-C3 on the surface of cells to examine the specific response superiority between C2 and C3 peptide antigenic epitopes and anti-core antibodies in serum of HCV positive patients ELISA was performed using the sera of 10 HCV infected patients. As shown in Example 6, 10 chronically infected patients' serum was diluted 1: 1000 in a microtiter plate coated with cells expressing each protein, and the anti-human Ig in which ALP (alkaline phosphatase) was bound was reacted. After binding by diluting to: 5000, the substrate was added and the degree of color development was compared by expressing the degree of color development of wells containing cells expressing only INC protein as a limit value (FIG. 8).

FIG. 8 shows the reactivity of major HCV core antigenic epitopes (c22p, C2 and C3) with core specific antibodies in serum of HCV infected patients. In comparison, C2 and C3 antigenic epitopes are sensitively reacted with anti-core antibodies in patient serum to show the superiority of these antigenic epitopes. The parenthesis under the patient number indicates the genotype of HCV infected and the number of copies of the viral gene RNA.

Antigen-antibody reactions using enzyme immunoassay were performed on sera of 10 infected patients using C2 and C3 antigenic epitopes that can sensitively detect newly detected HCV core antibodies and antigenic epitopes c22p that have been reported and used. As a result, it was confirmed that C2 (30%) and C3 (70%) were able to detect anti-HCV core antibodies more sensitively than the core antigenic epitope c22p currently used (FIG. 8).

The present invention has developed an HCV core antibody recognition epitope that reacts with antibodies more sensitively than the antigens used by microbial cell surface expression technology. Can be used effectively.

Simple modifications or variations of the present invention can be readily used by those skilled in the art, and all such modifications or changes can be regarded as being included in the scope of the present invention.

<110> GenoFocus <120> Core Antigenic Epitope for Diagnosis of Herpatitis C Virus          Antibody <160> 21 <170> KopatentIn 1.71 <210> 1 <211> 191 <212> PRT <213> Hepatitis C virus <220> <221> PEPTIDE (222) (10) .. (53) <223> HCV core antigenic epitope (c22p) <220> <221> PEPTIDE (222) (38) .. (53) <223> C1 <220> <221> PEPTIDE (222) (26) .. (53) <223> C2 <220> <221> PEPTIDE (222) (1) .. (38) <223> C3 <220> <221> PEPTIDE <222> (101) .. (121) <223> C4 <220> <221> PEPTIDE (222) (38) .. (75) <223> C5 <220> <221> PEPTIDE (222) (1) .. (26) <223> C6 <220> <221> PEPTIDE (222) (1) .. (75) <223> C7 <220> <221> PEPTIDE (222) (38) .. (121) <223> C8 <400> 1 Met Ser Thr Asn Pro Lys Pro Gln Arg Lys Thr Lys Arg Asn Thr Asn   1 5 10 15 Arg Arg Pro Gln Asp Val Lys Phe Pro Gly Gly Gly Gln Ile Val Gly              20 25 30 Gly Val Tyr Leu Leu Pro Arg Arg Gly Pro Arg Leu Gly Val Arg Ala          35 40 45 Thr Arg Lys Ala Ser Glu Arg Ser Gln Pro Arg Gly Arg Arg Gln Pro      50 55 60 Ile Pro Lys Ala Arg Arg Pro Glu Gly Arg Ala Trp Ala Gln Pro Gly  65 70 75 80 Tyr Pro Trp Pro Leu Tyr Gly Asn Glu Gly Leu Gly Trp Ala Gly Trp                  85 90 95 Leu Leu Ser Pro Arg Gly Ser Arg Pro Ser Trp Gly Pro Thr Asp Pro             100 105 110 Arg Arg Arg Ser Arg Asn Leu Gly Lys Val Ile Asp Thr Leu Thr Cys         115 120 125 Gly Phe Ala Asp Leu Met Gly Tyr Ile Pro Leu Val Gly Ala Pro Leu     130 135 140 Gly Gly Ala Ala Arg Ala Leu Ala His Gly Val Arg Val Leu Glu Asp 145 150 155 160 Gly Val Asn Tyr Ala Thr Gly Asn Leu Pro Gly Cys Ser Phe Ser Ile                 165 170 175 Phe Leu Leu Ala Leu Leu Ser Cys Leu Thr Ile Pro Ala Ser Ala             180 185 190 <210> 2 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> PCR primer for preparing pT7INC-core <400> 2 gattgggggc gacactccac c 21 <210> 3 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> PCR primer for preparing pT7INC-core <400> 3 ggaattcctt aagcggaggc tgggatggtc 30 <210> 4 <211> 33 <212> DNA <213> Artificial Sequence <220> <223> PCR primer for preparing pT7INC-C1 <400> 4 cgggatccgc gcaggggccc caggttgggt gtg 33 <210> 5 <211> 31 <212> DNA <213> Artificial Sequence <220> <223> PCR primer for preparing pT7INC-C1 <400> 5 ggaattcctt aggaagtctt cctagtcgcg c 31 <210> 6 <211> 33 <212> DNA <213> Artificial Sequence <220> <223> PCR primer for prepring pT7INC-C2 <400> 6 cgggatccgg gcggtggtca gatcgttggt gga 33 <210> 7 <211> 31 <212> DNA <213> Artificial Sequence <220> PCR primers <400> 7 ggaattcctt aggaagtctt cctagtcgcg c 31 <210> 8 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> PCR primer for preparing pT7INC-C3 <400> 8 gattgggggc gacactccac c 21 <210> 9 <211> 34 <212> DNA <213> Artificial Sequence <220> <223> PCR primer for prepring pT7INC-C3 <400> 9 ggaattctta cggcaacagg taaactccac caac 34 <210> 10 <211> 33 <212> DNA <213> Artificial Sequence <220> <223> PCR primer for prepring pT7INC-C4 <400> 10 cgggatcccc gtggctcccg gcctagttgg ggc 33 <210> 11 <211> 31 <212> DNA <213> Artificial Sequence <220> <223> PCR primer for prepring pT7INC-C4 <400> 11 ggaattcctt aacccaagtt acgcgaccta c 31 <210> 12 <211> 33 <212> DNA <213> Artificial Sequence <220> <223> PCR primer for prepring pT7INC-C5 <400> 12 cgggatccgc gcaggggccc caggttgggt gtg 33 <210> 13 <211> 34 <212> DNA <213> Artificial Sequence <220> <223> PCR primer for prepring pT7INC-C5 <400> 13 ggaattctta ccaggcccta ccctcgggct ggcg 34 <210> 14 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> PCR primer for prepring pT7INC-C6 <400> 14 gattgggggc gacactccac c 21 <210> 15 <211> 35 <212> DNA <213> Artificial Sequence <220> <223> PCR primer for prepring pT7INC-C6 <400> 15 ggaattctta cgggaacttg aacgtcctgt gggcg 35 <210> 16 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> PCR primer for prepring pT7INC-C7 <400> 16 gattgggggc gacactccac c 21 <210> 17 <211> 34 <212> DNA <213> Artificial Sequence <220> <223> PCR primer for prepring pT7INC-C7 <400> 17 ggaattctta ccaggcccta ccctcgggct ggcg 34 <210> 18 <211> 33 <212> DNA <213> Artificial Sequence <220> <223> PCR primer for prepring pT7INC-C8 <400> 18 cgggatccgc gcaggggccc caggttgggt gtg 33 <210> 19 <211> 31 <212> DNA <213> Artificial Sequence <220> <223> PCR primer for prepring pT7INC-C8 <400> 19 ggaattcctt aacccaagtt acgcgaccta c 31 <210> 20 <211> 29 <212> DNA <213> Artificial Sequence <220> <223> PCR primer for prepring pT7INC-C9 <400> 20 cgggatccca aaaccaaacg taacaccaa 29 <210> 21 <211> 31 <212> DNA <213> Artificial Sequence <220> <223> PCR primer for prepring pT7INC-C9 <400> 21 ggaattcctt aggaagtctt cctagtcgcg c 31

Claims (20)

C2 which is the amino acid sequence of 26 to 53 in SEQ ID NO: 1, C3 which is the amino acid sequence of 1 to 38 in SEQ ID NO: 1, C6 which is the amino acid sequence of 1 to 26 in SEQ ID NO: 1, and the amino acid sequence of 1 to 75 in SEQ ID NO: 1 Antigenic epitope for detection of HCV selected from the group consisting of C7. An oligonucleotide encoding the epitope of claim 1. A fusion protein in which two or more epitopes of the same epitope are fused or two or more different epitopes are fused. The cell surface expression vector containing the gene of the oligonucleotide and secretion induction protein of Claim 2, and the epitope expressed by the said oligonucleotide is comprised so that expression may be induced in the cell surface by the said secretion induction protein. The expression vector according to claim 4, wherein the secretory inducing protein is an ice nucleus active protein or part thereof. 6. The expression vector according to claim 5, wherein the ice-nucleus active protein is derived from Pseudomonas Schringe. 6. The expression vector according to claim 5, wherein a part of the ice nucleus activating protein is DNA encoding a cell attachment site N-terminal and a site C-terminal secretion to the outer membrane and a target function. Bacteria transformed with the expression vector of claim 4. A method of expressing the antigenic epitope of claim 1 on the surface of a bacterium, comprising culturing the bacterium of claim 8. A bacterium produced by the method of claim 9, wherein the antigenic epitope of claim 1 is expressed on the surface. delete A cell surface expression vector selected from the group consisting of pT7INC-core, pT7INC-c22p, pT7INC-C2, pT7INC-C3, pT7INC-C6 and pT7INC-C7. Bacteria transformed with the expression vector of claim 12. A method for expressing an antigenic protein or fragment thereof on the surface of a bacterial cell, comprising culturing the bacterium of claim 13. delete delete delete A kit for detecting HCV, comprising the epitope of claim 1, the fusion protein of claim 3, or the bacterium of claim 10. A composition for detecting HCV, comprising the epitope of claim 1, the fusion protein of claim 3, or the bacterium of claim 10 as an active ingredient. A primer pair selected from the group consisting of SEQ ID NO: 6 and SEQ ID NO: 7, SEQ ID NO: 8 and SEQ ID NO: 9, SEQ ID NO: 14 and 15, and SEQ ID NO: 16 and SEQ ID NO: 17.

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