KR20120103882A - Antigen for detecting of classical swine fever virus, antibody detecting method and test kit using thereof - Google Patents

Abstract

PURPOSE: A fusion peptide which is specific to an antibody for classical swine fever virus and a kit containing the same are provided to enhance specificity and sensitivity. CONSTITUTION: A fusion peptide which is specific to an antibody for classical swine fever virus essentially contains amino acid sequences of sequence number 20 at 414-421th site from N-terminal, sequence number 21 at 423-437th site, and sequence number 22 at 463-471th site and also repetitively contains antigen-derived peptides of 58-68 serial amino acids.

Description

Antigen for detecting swine fever virus, antibody detection method using the same, and detection kit including the same {ANTIGEN FOR DETECTING OF CLASSICAL SWINE FEVER VIRUS, ANTIBODY DETECTING METHOD AND TEST KIT USING THEREOF}

The present invention relates to a fusion peptide specific for an antibody against swine fever virus, and a detection kit for swine fever virus infection comprising the same.

Swine fever virus (CSFV) is an RNA virus belonging to the genus Flaviviridae and Pestivirus . Its size is about 40 ~ 50nm, and the swine fever virus gene is positive sense single strand RNA, which is about 12.3 kb in size. The code synthesizes about 3900 amino acids and constitutes a viral structural protein. The swine fever virus protein consists of Npro, Capsid, Erns, E1, E2, p7, NS2, NS3, NS4A, NS4B, NS5A and NS5B. Swine fever virus is classified into three genotypes by comparing the 5 ′ non-coding region (NCR), E2, and NS5B gene sequences.

Pigs infected with swine fever virus develop antibodies against ERNS, E2, and NS3. Of these viral proteins, the E2 protein constitutes the envelope of CSFV and is known as a major protective antigen involved in producing antibodies that strongly neutralize the virus. Swine fever virus is infected by feces, urine, tears, and runny nose of infected pigs, but also by mechanical agents such as people, shoes, clothing, and utensils. Since wild viruses are also found in wild boars, wild boar areas are in danger of developing, and when infected, acute forms of fever include high fever, skin redness, appetite deficiency, constipation, diarrhea, white blood cell reduction, posterior palsy and mortality. In particular, when pregnant sows are infected by the fetus through the placenta, reabsorption, miscarriage, stillbirth, etc. appear according to age, and even when delivered, they die or contract after several days of delivery. The incubation period is usually 6-11 days or 20-30 days (up to 40 days according to the International Water Organization). Therefore, it is important to diagnose early and prevent the spread because it can spread throughout the country before clinical symptoms appear.

As of 2009, advanced livestock countries such as the United States and Japan have been selected as clean areas by the International Water Bureau due to thorough protection against swine fever, but have also occurred in Asian countries, including China, and some Europe, including Bulgaria, Romania, and Hungary. have. In some European countries, where swine fever infections are not found in livestock pigs, infections are found in wild boars, which are not safe for swine fever and require rapid diagnostic tools.

Currently, in Korea, immunization with LOM strain, a swine fever-purifying live venom virus, is performed as a live venom vaccine. As a result, vaccine vaccines and field reading virus discrimination kits have been developed and used by two companies (CEDI and CHECKIT), but they have been reported to show inconsistent test results depending on the genotype of the virus. Is required.

Currently, Korea is vaccinating live vaccines made with LOM strain, a swine fever purified live poison virus, but plans to commercialize the recombinant marker vaccine using ERNS protein to eradicate swine fever. However, when a recombinant marker vaccine is used, a test method for distinguishing a vaccine from an outdoor infection antibody has not been established.

Accordingly, the present invention provides an antigen peptide specific to the swine fever virus, in particular, the antibody against the ERNS protein of the virus, and a detection kit comprising the same, and the antigen peptide and / Or using the detection kit to detect whether the swine fever virus infection and specific antibodies against swine fever virus, as well as providing a method for discriminating easily and accurately purified live poison vaccine strain and recombinant marker vaccine strain or outdoor strain For the purpose of

In order to solve the above problems, the present inventors detect the ERNS antibody against the vaccine strain administered for the prevention of Swine Fever Virus (CSFV), purified live vaccine vaccine, recombinant marker vaccine strain, or outdoor strain As a result of research efforts to establish a method for accurately discriminating against humans, we have developed an antigen protein that specifically binds to ERNS antibody, one of antibodies induced by Swine fever virus infection, and a rapid detection kit containing the same. The present invention has been completed. As a result, not only the vaccine and field strain differential diagnosis of swine fever virus but also all genotypes of swine fever virus can be diagnosed.

Accordingly, the present invention provides an antigen-derived peptide which is an immunodominant epitope present in the ERNS protein of swine fever virus.

In addition, the present invention is essential to the amino acid sequence of SEQ ID NO: 20 located at 414 to 421th from SEQ ID NO: 1, SEQ ID NO: 21 located at 423 to 437, and SEQ ID NO: 22 located at 463 to 471th from the N-terminus It provides a fusion peptide specific for the antibody against the swine fever virus, including as repeated in series two or more times the antigen-derived peptide consisting of 58 to 68 consecutive amino acids, including.

In addition, the present invention is essential to the amino acid sequence of SEQ ID NO: 20 located at 414 to 421th from SEQ ID NO: 1, SEQ ID NO: 21 located at 423 to 437, and SEQ ID NO: 22 located at 463 to 471th from the N-terminus Including a contiguous peptide consisting of 58 to 68 consecutive amino acids, or comprising a repeated two or more times in series the peptide, it provides a composition for detecting swine fever virus infection.

In another aspect, the present invention provides a detection kit of swine fever virus infection comprising the composition for detecting swine fever virus infection.

In another aspect, the present invention provides a method for discriminating the vaccine strain or outdoor strain of swine fever virus using the swine fever virus infection detection composition or the detection kit.

Also,

Preparing a sample;

The amino acid sequence of SEQ ID NO: 20 located at 414 to 421th from SEQ ID NO: 1, SEQ ID NO: 21 located at 423 to 437th, and SEQ ID NO: 22 located at 463 to 471th of the amino acid sequence of SEQ ID NO: 1 are essential to the specimen. Applying an antigen-derived peptide consisting of 58 to 68 consecutive amino acids, or a fusion peptide comprising two or more times repeated in series; And

Examining the antigen-antibody reaction of the sample and the fusion peptide

Provided, the antibody detection method for swine fever virus.

The present invention also provides a diagnostic method for determining whether the swine fever virus infection or vaccine prescription using the swine fever virus infection detection composition or the detection kit.

Hereinafter, the present invention will be described in more detail.

Pigs infected with the swine fever virus produce antibodies against the structural proteins ERNS and E2 and the nonstructural protein NS3. The membrane glycoprotein E2 is a representative immunogenic protein of swine fever virus. Another membrane glycoprotein, ERNS, has RNase activity. The RNase activity of ERNS is responsible for virus replication and continuous infection in the host. (Edwards et al., 1991; Vet. Microbiol. 29: 101-108, Weiland et al., 1992; J. Virol. 66: 3677-3682, Hulst et al., 1998; J. Virol 72 (1): 151-157).

In Korea, the immunization of the LOM strain, a swine fever purified live venom virus, is performed as a live venom vaccine, but in the case of live venom virus, side effects of activating the virus due to the variation of the vaccine strain in the inoculated pigs are occurring. For this reason, the use of marker vaccines without E2 vaccine or ERNS antigens is already recommended in foreign countries. Currently, the most widely used vaccine is a subunit vaccine using purified E2 protein as an immunogen (Bouma et al., 1999; Vet. Microbiol. 66: 101-114). However, since these four vaccines show immunity against a single protein, there is a disadvantage in that the immune response to protecting inoculated pigs from fever virus infection may be weaker than that of live vaccines (Hulst et al., 1993; J. Virol). 67: 5435-5442, Meyers et al., 1996; J. Virol. 70: 1588-1595, Ruggli et al., 1996; J. Virol. 70: 3478-3487). To compensate for this, a recombinant marker vaccine was introduced. For example, a vaccine that suppresses infectivity caused by side effects while maintaining a function as a vaccine by making a mutant vaccine strain in which a part of the ERNS structural protein is deleted or replacing a heterologous ERNS is available. Are used (Widjojoatmodjo et al., 2000; J. Virol. 74: 2973-2980, Song et al., Republic of Korea Patent Publication 10-2010-0121288).

The present inventors select the optimal target of the effective antigen by analyzing the nucleic acid sequence and amino acid sequence of the target gene based on bioinformatics analysis technology to produce a recombinant protein of the important effective antigen from the ERNS structural protein of the swine fever virus An immunodominant epitope in the ERNS protein of porcine fever virus was designed.

Accordingly, the present invention provides an ERNS antigen-derived peptide which is an immunodominant epitope present in the ERNS protein of swine fever virus.

The antigen-derived peptide is designed to be diagnosed regardless of all genotypes of swine fever virus as well as differential diagnosis of vaccine and outdoor strains of swine fever virus, and preferably, N- in the amino acid sequence of swine fever virus of SEQ ID NO: 1 It may consist essentially of 58 to 68 contiguous amino acids, including essentially the amino acid sequence of SEQ ID NO: 20 located at 414 to 421th, SEQ ID NO: 21 located at 423 to 437, and SEQ ID NO: 22 located at 463 to 471 from the end. have.

The amino acid sequences of SEQ ID NOs: 20, 21, and 22, which are essentially included in the antigen-derived peptide, are effective target antigens present in the ERNS structural protein located 268-494th from the N-terminus of the amino acid sequence of the swine fever virus of SEQ ID NO: 1. As the domain, amino acids other than the essential amino acid sequences of the amino acid sequence of the antigen-derived peptide may be substituted.

The antigen-derived peptide may be composed of 58 to 68 consecutive amino acids, more preferably 58 to 62, preferably including the amino acid sequence of SEQ ID NO: 2.

Most preferably, the antigen-derived peptide may be a peptide (ERNS-F1) consisting of the amino acid sequence of SEQ ID NO: 2.

The amino acid sequence of SEQ ID NO: 2 may be encoded by the nucleotide sequence of SEQ ID NO: 3.

The present invention is also a fusion peptide comprising the ERNS antigen-derived peptide repeatedly, specifically SEQ ID NO: 20, 423 to 437, located at 414 to 421th from the N-terminus of the amino acid sequence of the swine fever virus of SEQ ID NO: 1 At least two, preferably two to five, antigen-derived peptides consisting essentially of 58 to 68 consecutive amino acids comprising essentially the amino acid sequence of SEQ ID NO: 21 and SEQ ID NO: 22 located at 463-471 Provided, a fusion peptide specific for an antibody against swine fever virus, which is included repeatedly.

A fusion peptide specific for an antibody against swine fever virus, wherein the antigen-derived peptide is repeatedly included two or more times in series, is preferably an amino acid of SEQ ID NO: 4 in which the antigen-derived peptide is repeatedly included two times in series. A fusion peptide consisting of a sequence (118 amino acids) (ERNS-F2), or a fusion peptide (ERNS) consisting of an amino acid sequence of SEQ ID NO: 6 (178 amino acids) in which the antigen-derived peptide is repeated three times in series -F3).

The amino acid sequence of SEQ ID NO: 4 may be encoded by the nucleotide sequence of SEQ ID NO: 5, the amino acid sequence of SEQ ID NO: 6 may be encoded by the nucleotide sequence of SEQ ID NO: 7.

The fusion peptide may preferably be a recombinant fusion peptide prepared by a recombinant method using the antigen-derived peptide, wherein the ERNS antigen-derived peptides ERNS-F1, ERNS-F2, and ERNS-F3 are preferably directly synthesized or Or a genomic gene using a polymerase chain reaction (PCR) method or the like. In a specific embodiment, to obtain the gene of ERNS-F1, a specific primer pair from the template genomic gene can be used to amplify the target fragment of the ERNS-F1 gene by PCR to prepare a complete antigen protein gene.

Gene production method of the antigenic protein comprises the synthesis of a series of oligonucleotides having a sequence overlapping with the complementary end of the nucleic acid to be amplified. The oligonucleotide may be one containing several kinds of restriction enzyme sites for cloning into an expression vector. In heat cooling, the ends are used as primers for the amplification of complementary strands, and the amplification products are amplified by extra external primers.

The fusion peptide captures only ERNS antibodies in a recombinant form in which immunodominant epitopes present in the ERNS protein of porcine fever virus are continuously or discontinuously serially fused, and an assay strip prepared using the same The rapid immunochromatography method can differentially detect the presence or absence of ERNS antibody derived from CSFV open or live vaccine or E2 vaccine in the sample.

Accordingly, the present invention is essential to the amino acid sequence of SEQ ID NO: 20 located at 414 to 421th from SEQ ID NO: 1, SEQ ID NO: 21 located at 423 to 437, and SEQ ID NO: 22 located at 463 to 471 of the amino acid sequence of SEQ ID NO: 1 Provided with an antigen-derived peptide consisting of 58 to 68 consecutive amino acids, or comprising two or more times, preferably two to five times in series, comprising a continuous swine fever virus infection detection composition. .

The present invention also provides an assay strip for detecting ERNS antibody comprising the composition for detecting swine fever virus infection.

The present invention also provides a detection kit for swine fever virus infection comprising the composition for detecting swine fever virus infection.

The infection detection composition and / or the detection kit may be used to detect specific antibodies against swine fever virus infection and swine fever virus. It can be discriminated quickly and simply and can be confirmed whether swine fever virus infection and antibody production with high specificity and sensitivity, and thus can be effectively used for early diagnosis, prevention and treatment of swine fever virus.

In detail, the detection kit detects the presence or absence of an antibody against the ERNS antigen present in the sample, so that the positive may be determined by a swine fever virus infection and / or a live venom vaccine, and the negative may be determined by a marker vaccine. .

The kit may preferably be to detect an ERNS antibody against infection of porcine fever virus by using an antigen-antibody reaction with a sample and the infection detection composition, that is, the antigen-derived peptide or the fusion peptide. It may be an immunokit capable of detecting vaccine differentiation of swine fever virus.

The sample preferably includes, but is not limited to, fluids of a subject to be diagnosed, such as blood, serum, plasma, lymph, tissue fluid, or urine, tears, saliva, milk, vomiting, feces, etc. secreted into the body. .

The kit may be in the form of, but is not limited to, an enzyme immunoassay kit (ELISA), a blotting kit, an immunoprecipitation kit, an immunofluorescence kit, or an immune strip.

The kit may comprise one or more assay strips comprising a sample pad absorbing a sample and a membrane representing the detection result and an absorbent pad absorbing the sample developing solution, wherein the assay strip is preferably short and wide in length. It may have a shape having a length of.

The detection kit in the form of an immunostrip preferably has two or more assay strips, wherein any one of the assay strips comprises a membrane to which the fusion peptide is immobilized that can specifically bind to swine fever virus or ERNS antibody of a vaccine strain. And other assay strips may include membranes to which additional antigens or antibodies are immobilized.

The detection kit has a lower case for supporting one side of the analysis strip; And an upper case coupled to the lower case to cover the other surface of the assay strip, the upper case including a sample inlet corresponding to the sample pad, a result display window corresponding to the membrane, and a letter display corresponding to the absorbent pad. It may be included as (see FIGS. 3 to 6).

In order to detect swine fever virus by immunochromatography, the raw material for detecting swine fever virus antigen protein ERNS-F1 or ERNS-F2 or -F3 recombinant fusion antigen is adsorbed on a nitrocellulose membrane at a predetermined position. The antibody or antigen for each can be conjugated to and dried on a pad. In addition, the dried gold (gold) conjugate pad and the pad to apply the sample may be overlaid on the nitrocellulose membrane and the absorbent pad for absorption in the opposite position (Figs. 5 and 6).

Membrane that can be used for the production of the assay strip may be used as a material of a conventional diagnostic strip, for example, may be one selected from various synthetic polymers such as nitrocellulose, cellulose, cellulose acetate, polyethylene, etc. .

In addition, the kit may further include a detection reagent for detecting a sample hybridized with the antibody, and the detection reagent may be externally identified through the naked eye or other apparatus to detect the presence of an analyte to be tested. Certain labeled reagents, auxiliary specific binding members, and / or components of the signaling system.

The labeling reagent, i.e., a labeled detection reagent, may be one well known to those skilled in the art, and the label may be, for example, a catalyst, an enzyme (for example, phosphatase, peroxidase, etc.). More specific examples include basic phosphatase and red pepper peroxidase used in combination with enzyme substrates, enzyme substrates (eg, nitrobluetetrazolium, 3,5 ', 5,5'-tetranitro Benzidine, 4-methoxy 1 naphthol, 4 chloro 1 naphthol, 5-bromo-4-chloro-3-indolyl phosphate, dioxetane as chemiluminescent enzyme substrate and inducers and analogs thereof), fluorescent compounds ( For example, fluorescein, phycobiliprotein, rhodamine, derivatives and the like), chemiluminescent compounds, metal sol, dye sol, particulate latex (Particulate latex), Color indicator (Col or indicator), color matter contained in liposomes, chemiluminescent compounds, metal sol, dye sol, particulate latex, color indicator, liposomes It may be one or more selected from color matter, carbon sol and non-metal sol such as selenium, but is not limited thereto.

Labeling reagents that may be included in the control reagent may be applied in the same manner as the label in the detection reagent.

Examples of the auxiliary specific binding member do not require special limitation, but may be, for example, one selected from avidin, biotin, FITC, anti-FITC antibody, mouse immunoglobulin, and anti mouse immunoglobulin antibody.

4, the test line 221 having at least one stationary phase selected from antibodies or heptenes derived from porcine fever virus, and the control line 222 for determining whether the kit operates normally are the membranes 22. An analysis strip 2 provided in a predetermined area on the? A result display window for protecting the assay strip 2 from various contaminants, and for observing the reaction result at the test line 221 on the test strip 221 and the control line 222 on at least a sample inlet 41 for introducing a sample ( 42) as an immunoassay device (1) equipped with may be used for differential diagnosis of swine fever virus vaccine.

The kit facilitates the discrimination of swine fever virus LOM strains of purified live read vaccine vaccines and gene synthesis marker vaccine strains as well as field strains. Therefore, it can be effectively used for the prevention and treatment of swine fever through the serological differentiation of the swine fever prevention vaccine and the outdoor strain through the present invention.

4 is an exploded perspective view of an immunoassay device according to an embodiment of the present invention. Referring to FIG. 4, the immunoassay device 1 includes an analysis strip 2 that absorbs a sample and displays a detection result, and a lower case 3 and an upper case 4 which are combined with each other to embed the analysis strip 2. It includes.

The lower case and the upper case may contain one assay strip to enable only one disease test at a time (not shown). In addition, the lower case and the upper case may be configured to include three or more assay strips to test for a plurality of diseases (not shown).

Preferably, the lower case 3 and the upper case 4 incorporate a pair of assay strips 2 to enable testing for two diseases at a time. In addition, for the sake of convenience, the drawing illustrates a structure in which a pair of analysis strips 2 are embedded, and in the general description, one analysis strip 2 will be mainly described in order to avoid duplication.

Briefly described with respect to the pair of analysis strips 2, the lower case 3 includes a first strip support 3A and a second strip support 3B that support the pair of analysis strips 2. Accordingly, the upper case 4 has a first strip counterpart 4A and a second strip counterpart corresponding to the pair of analysis strips 2, that is, the first strip support 3A and the second strip support 3B, respectively. (4B).

The first strip support 3A and the first strip counterpart 4A support and embed both surfaces (lower surface and upper surface in FIG. 4) of one analysis strip 2. The second strip support 3B and the second strip counterpart 4B support and embed both surfaces (lower surface and upper surface in FIG. 4) of the other analysis strip 2.

The assay strip 2 includes a sample pad 21 for absorbing a sample, a membrane 22 representing a detection result, and an absorbent pad 23 for absorbing a sample's development liquid. The membrane 22 includes a test line 221 reacting by a sample and a control line 222 which is a standard for disease analysis by comparing the test line.

The analysis strip 2 also has a width W24 of the short side 24 and a length L25 of the long side 25 so that it can be fixed between the strip support 3A and the strip counterpart 4A. 1 strip support 3A and 1st strip counterpart 4A).

Additional sample development solution was added dropwise (for example 40 μl (1 drop)) and capillary phenomenon on the nitrocellulose membrane 22 was carried out by reacting the swine fever virus ERNS antibody present in the sample with each antibody or antigen attached to the gold particles. Developed by. A specific ERNS recombinant antigen capable of detecting swine fever virus ERNS antibody is adsorbed at a predetermined position on the test line 221 of the membrane, so that when an infectious antibody of the infectious agent is present in the sample, the antigen conjugated to the gold particle or Viral Antibodies Responding to Antibodies React with antigens adsorbed on the membrane to form purple (red) bands of gold particle color at the site. Anti-mouse IgG was adsorbed at the control line 222 so that gold particles adsorbed with mouse IgG always reacted regardless of the presence of viral antigens in the sample to show violet (red) bands (FIGS. 4 and 5). . The unreacted content penetrates the absorbent pad so that the membrane in the inspection window appears clean white, making the bands easily readable. In addition, if no swine fever virus ERNS-specific antibody is present in the sample, a purple or red band is formed only at the control region of the strip (FIG. 3).

The lower case 3 is formed to support one surface, for example, the lower surface of the analysis strip 2 embedded therein. The upper case 4 is formed to cover the other side of the analysis strip 2, for example, the upper surface, coupled to the lower case 3 and embedded therein.

The upper case 4 has a sample inlet 41 formed corresponding to the sample pad 21, a result display window 42 formed corresponding to the membrane 22, and a character formed corresponding to the absorbent pad 23. It includes the indicator port 43.

The present invention provides a method for discriminating the vaccine strain or outdoor strain of swine fever virus using the detection kit.

The vaccine strain may be preferably a purified live read vaccine strain or a recombinant marker vaccine strain, but is not limited thereto and may be applied to all types of swine fever vaccines that do not produce ERNS antibodies of swine fever virus.

In another aspect, the present invention comprises the steps of preparing a sample; Applying a fusion peptide specific for the antibody against the swine fever virus to the sample; And it provides an antibody detection method for swine fever virus, comprising the step of examining the antigen-antibody reaction of the sample and the fusion peptide.

In another aspect, the present invention provides a diagnostic method for determining whether or not swine fever virus infection or vaccine prescription using the swine fever virus infection detection composition or detection kit of the present invention.

The diagnostic method preferably includes the steps of (1) injecting a sample into a region of the assay strip; (2) combining the detection reagent (gold (gold) conjugate) with a predetermined label with the analyte in the sample to form a complex; (3) deploying the composite to the membrane; And (4) monitoring changes in the appearance of the reaction site having at least one stationary phase selected from antibodies or heptenes obtained from the swine fever virus infection or vaccine in a predetermined region on the membrane. It may include determining whether or not a prescription.

The diagnostic method includes a sandwich assay or a competition assay. For example, in the immunoassay of the present invention, after fixing the fusion peptide according to the present invention to the membrane in the immune strip and reacting a sample from the serum of the animal, the labeled secondary antibody against the antibody contained in the sample is It can also be analyzed by the reacted sandwich method.

Antigen-derived peptides, fusion peptides, detection kits of the present invention as described above, a method for discriminating a vaccine strain or an outdoor strain of swine fever virus using the same, and an antibody detection method for swine fever virus, etc. Monitoring of antibodies and differential testing of infectious antibodies in marker-vaccine pigs lacking ERNS are possible. These results are expected to contribute to the eradication of swine fever.

The present invention provides a fusion peptide specific for a swine fever virus, in particular, an antibody against an ERNS protein of the virus, and a detection kit comprising the same, and whether the swine fever virus is infected using the fusion peptide and / or the detection kit, and In addition to detecting specific antibodies to swine fever virus, we also provide a way to easily differentiate between purified live read vaccine vaccines and gene marker marker vaccines and outdoor strains, providing rapid, simple, high specificity and sensitivity to swine fever viruses. Differential diagnosis is possible for vaccine strains and field strains of swine fever virus, including infection and antibody production, and can be used very usefully, regardless of all genotypes of swine fever virus. Accordingly, the present invention can effectively contribute to the early diagnosis, prevention and treatment of swine fever virus.

1 shows antigenicity test results through dot-Immunoassay of recombinant ERNS-F2 / -F3 antigenic protein according to an embodiment of the present invention.
Figure 2 shows the results of antigenicity test through direct enzyme immunoassay of recombinant ERNS-F2 / -F3 antigen protein according to an embodiment of the present invention.
Figure 3 is a photograph of an example of the reaction result after the application of the sample of the detection kit of swine fever virus infection according to an embodiment of the present invention.
Figure 4 schematically shows an exploded perspective view of the detection kit of swine fever virus infection according to an embodiment of the present invention.
Figure 5 schematically shows the structure of the strip constituting the detection kit for swine fever virus infection according to an embodiment of the present invention.
Figure 6 is a photograph of a detection kit of swine fever virus infection according to an embodiment of the present invention.
7 is a graph showing the results of Predicted transmembrane segments for sequence of ERNS.
FIG. 8 is a graph of the hydrophilic (FIG. 8A) / hydrophobic (FIG. 8B) parameters of mature ERNS protein excluding transmembrane fragment sites. FIG.
9A-9C show antigenic peptides of the ERNS effective antigenic sites of BVDV (KD26-1) (FIG. 9A), CSFV LOM strain (FIG. 9B), and CSFV SW03 strain (FIG. 9C), respectively. .
10A and 10B are FPLC results graphs for ERNS-F2 (FIG. 10A) and purified electrophoresis pictures of ERNS-F2 (FIG. 10B: red square in FIG. 10A).
11A and 11B are FPLC results graphs for ERNS-F3 (FIG. 11A) and purified electrophoresis images of ERNS-F2 (FIG. 11B: red square in FIG. 11A).
Figure 12 shows antigenic peptides and effective target antigen domains (candidate 1, candidate 2, and candidate 3) of ERNS effective antigenic sites of BVDV KD26-1, CSFV LOM, CSFV SW03 (red squares) ).
Description of the Related Art
1: immunoassay device
2: assay strip
21: sample pad 22: membrane
221: inspection line 222: control line
23: absorbent pad 24: short side
25: long side
3: lower case
3A, 3B: first and second strip support
31: short-side fixing part
32: long side fixing part 33: lower blocking part
4: upper case
4A, 4B: first and second strip counterpart
41: sample inlet 42: result display window
43: character display port

Hereinafter, the swine fever diagnosis system and its manufacturing method according to the present invention will be described in more detail with reference to Examples. However, these examples are only presented to understand the content of the present invention, but the scope of the present invention is not intended to be limited to these embodiments.

Example 1 : Swine Fever Virus  For differential diagnosis of vaccine antibodies ERNS -F2 and ERNS Preparation of F3 Antigen

A. Recombination ERNS -F1 Antigen Protein Design and Gene Production

(a) Analysis and selection of target ERNS structural proteins.

In order to produce recombinant proteins of important effective antigens from the ERNS structural protein of swine fever virus, the optimal nucleic acid and amino acid sequences of the target genes are analyzed based on bioinformatics analysis technology to secure specific antigenic and water-soluble proteins of the antigen. Targets of effective antigen were selected.

The amino acid sequence for the ERNS antigen protein for effective antigen analysis was derived from Bovine viral diarrhea virus (KD26-1) and pigs belonging to the Flaviviridae (Pestivirus ) from the National Veterinary Research and Quarantine Service. LOM strain for fever virus vaccine and SW03 strain were used.

Analysis of the presence of the transmembrane segment of the translation product of the target gene revealed that the amino acid sequence was analyzed for the exclusion of the transmembrane segment. A total of 26 amino acids were analyzed as transmembrane proteins. Predicted transmembrane segments for sequence results graph is shown in FIG.

The transmembrane fragment site was excluded from the translation product of the target gene and analyzed to determine the hydrophilic and hydrophobic sites of the amino acid sequence constituting the ERNS target protein. A graph of the hydrophilic / hydrophobic parameters of the ERNS protein excluding transmembrane fragments is shown in FIG. 8 (hydrophilic parameters: FIG. 8A / hydrophobic parameters: FIG. 8B).

As a result of analyzing the antigenic peptide of ERNS to determine the effective antigenic position in the translation product of the target gene, ERNS of bovine viral diarrheal virus has 11 antigenic peptides at the effective antigenic site and swine fever. The ERNS of the virus LOM strain was analyzed with 11 antigenic peptides in the effective antigenic region and the ERNS of the swine fever virus SW03 strain with 10 antigenic peptides in the effective antigenic region, and are shown in FIGS. 9A, 9B, and 9C, respectively.

Based on the above results, the design of an effective antigen of ERNS is based on the analysis of profiles of antigenic peptides and amino acid sequences in common from the three viral strains except for transmembrane fragments. domain) (candidate 1 (SEQ ID NO: 20), 2 (SEQ ID NO: 21), and 3 (SEQ ID NO: 22) of FIG. 12) partially to maximize the antigenicity and solubility (water solubility) of the protein. Designed.

174 bp oligonucleotide (SEQ ID NO: 3) was synthesized in Bionics, Korea to secure the gene of ERNS-F1, which was designed to include the effective target antigen domain (SEQ ID NOS: 20-22). The synthesized oligonucleotide (SEQ ID NO: 3) was inserted between the multiple cloning sites BamHI and EcoRI in the expression vector pET23a (Novagen) using restriction enzymes BamHI (NEB) and EcoRI (NEB), which were pET23a (+) ERNS Named -F1, the nucleotide sequence and amino acid sequence including the BamHI and EcoRI restriction enzyme site, and the oligonucleotide of SEQ ID NO: 3 are shown in SEQ ID NO: 8 and SEQ ID NO: 9, respectively.

Since the designed ERNS-F1 contains an effective target antigen domain, which is an antigenic peptide of the ERNS effective antigenic region common to the swine fever virus LOM strain and the swine fever virus SW03 strain, all of the genotypes of swine fever virus Regardless, the diagnosis is expected to be possible.

B. Recombination ERNS Preparation of -F2 / -F3 Antigen Protein Expression Vectors

(a) Experimental Material Preparation and Polymerase Chain Reaction Method

The pET23a (+) ERNS-F2 / -F3 antigen protein expression vector uses pET23a (+) ERNS-F1 (SEQ ID NO: 8) prepared as a template, and the oligonucleotide for gene amplification is Bionics, Korea. It was synthesized in (Table 1).

Oligonucleotides Used to Prepare ERNS Antigens SEQ ID No. Name Sequence (5` → 3`) Other 10 ERNS-F2 sense cg gAATTC GCAGGTACAGTTATAGAG EcoRl 11 ERNS-F2 anti-sense c aAGCTT GAGCCAAGATGTTACCCT Hindlll 12 ERNS-F3 sense ccc aAGCTT GCAGGTACAGTTATAGAG Hindlll 13 ERNS-F3 anti-sense cc cTCGAG GAGCCAAGATGTTACCCT Xhol

The oligonucleotide was prepared containing a restriction site for cloning with the expression vector pET23a (+).

Polymerase chain reaction (50 ul volume) was set as follows. Pfu DNA polymerase (1U) (Promega) and 1X buffer, as well as 25 uM of each dNTP (dATP, dCTP, dGTP and dTTP) (Promega), 10 pmol of each oligonucleotide Sense primer (SEQ ID NOs: 10 and 12) And Anti-Sense primers (SEQ ID NOs: 11 and 13). The reaction was incubated at 95 ° C. for 5 minutes, amplified by circulating 27 seconds at 15 ° C. at 95 ° C., 30 seconds at 58 ° C., and 40 seconds at 72 ° C., and left at 72 ° C. for 2 minutes. The polymerase chain-product was separated from the gel after confirming that the size of the final product was consistent after loading on 10% agarose gel.

(b) Preparation of pET23a (+) ERNS-F2 Antigen Protein Expression Vector

External primers ERNS-F2 sense (SEQ ID NO: 10) and ERNS-F2 anti-sense (SEQ ID NO: 10) in the template pET23a (+) ERNS-F1 (SEQ ID NO: 8) to prepare a vector expressing an ERNS-F2 recombinant antigen protein 11), ERNS-F1 antigen-derived nucleic acid (PCR-ERNS-F2, SEQ ID NO: 14) having a total length of 174 bp (SEQ ID NO: 3) (58 amino acids: SEQ ID NO: 2) was obtained by a polymerase chain reaction method. Amplified. Thereafter, in the expression vector pET23a (+) ERNS-F1 prepared in Example 1A (a), EcoRI, which is a multiple cloning site of the 3 'terminal portion of the oligonucleotide of SEQ ID NO: 3, and a neighboring multiple cloning site, The amplified polymerase chain reaction product was inserted between HindIII using restriction enzymes EcoRI (NEB) and HindIII (NEB) to prepare an ERNS-F2 antigen protein expression vector containing two repeated ERNS-F1 antigen proteins. It was named pET23a (+) ERNS-F2. SEQ ID NO: 15 and SEQ ID NO: 16 show each restriction enzyme site, the nucleotide sequence including the amplified oligonucleotide, and the amino acid sequence, respectively.

(c) Preparation of pET23a (+) ERNS-F3 Antigen Protein Expression Vector

External primers ERNS-F3 sense (SEQ ID NO: 12) and ERNS-F3 anti-sense (SEQ ID NO: 12) in the template pET23a (+) ERNS-F1 (SEQ ID NO: 8) to prepare a vector expressing an ERNS-F3 recombinant antigen protein 13), the ERNS-F1 antigen-derived nucleic acid (PCR-ERNS-F3, SEQ ID NO: 17) having a total length of 174 bp (SEQ ID NO: 3) (58 amino acids: SEQ ID NO: 2) was obtained by a polymerase chain reaction method. Amplified. Thereafter, in the expression vector pET23a (+) ERNS-F2 prepared in Example 1A (b), HindIII, which is the multiple cloning site of the 3 'terminal portion of the oligonucleotide of SEQ ID NO: 3, and a neighboring multiple cloning site The amplified polymerase chain reaction product was inserted between XhoIs using restriction enzymes HindIII (NEB) and XhoI (NEB) to prepare an ERNS-F3 antigenic protein expression vector containing three repeated ERNS-F1 antigenic proteins. This was named pET23a (+) ERNS-F3. SEQ ID NO: 18 and SEQ ID NO: 19 show each restriction enzyme site, the nucleotide sequence including the amplified oligonucleotide, and the amino acid sequence.

(d) Cloning of the polymerase chain reaction product

Specifically, the amplified polymerase chain products for introduction of the polymerase chain reaction product into pET23a (+) ERNS-F1 by the above-mentioned method are respectively restricted to nucleic acid endonucleases EcoRI + HindIII (NEB). And serially connected to the vector pET23a (+) ERNS-F1, which was digested with HindIII + XohI (NEB) and gel-separated.

The serially linked binding products, pET23a (+) ERNS-F2 and pET23a (+) ERNS-F3, are used to transform E. coli DH5-alpha receptors (Invitrogens). The transformed cells were incubated overnight in a shaking orbital incubator at 37 ° C. in LB medium (MPbio) added with 100 ug / ml of empicillin. DNAs for minipreps were obtained by overnight incubation from colony medium, BamHI + HindIII (NEB) (pET23a (+) ERNS-F2 (SEQ ID NO: 15), 354 bases) and BamHI + XhoI (NEB) (pET23a (+) ERNS Certain clones were selected by digestion with -F3 (SEQ ID NO: 18), 534 base). In addition, sequencing confirmed the exact sequence of the cloning products (not shown).

C. Recombination ERNS Induction of Growth and Expression of Escherichia Coli Strains with -F2 / -F3 Antigen Protein Expression Vectors

Seeds of each E. coli clone selected in Example 1B were incubated overnight in a shaking orbital incubator at 37 ° C. in 50 ml of sterile LB medium (MPbio) added with 100 μg / ml of empicillin. 50 ml of inoculum was transferred from the seed to a flask containing 0.5 L of sterile LB (MPbio) with 100 μg / ml of ampicillin, incubated at 37 ° C. until they were medium-proliferated (OD value approximately 0.7). , pET23a (+) ERNS-F2 was incubated overnight at 25 ° C with 0.5mM IPTG (isopropylthiogalactoside), and pET23a (+) ERNS-F3 was incubated at 37 ° C for 4 hours with 0.5mM IPTG (isopropylthiogalactoside). It was. After the induction phase, cells were pelleted (7000 rpm, 10 min) in a centrifuge and harvested according to standard procedures. Pelletized cells were stored at -70 ° C until the next step. In order to analyze soluble and insoluble protein expression, the fusion protein was resuspended with Lysis-Equilibration buffer (50 mM NaH ₂ PO ₄ , 300 mM NaCl, 1 mM Immidazole, pH8.0), and the cells were subjected to sonication. By grinding. Cell-soluble centrifugation (13,000rpm, 30 minutes) was separated into water soluble and insoluble lysate and analyzed on 15% SDS-PAGE to determine whether the soluble protein was all insoluble protein.

D. Recombination ERNS Preparation of the -F2 / -F3 Antigen

Frozen cells obtained from Example 1C were prepared according to each POLYHISTIDINE-TAGGED protein purification standard procedure.

Each standard procedure is as follows.

All the cells that confirmed the expression were expressed as insoluble proteins, denatured, and then refolded. ERNS-F2 and ERNS-F3 proteins were resuspended with Lysis-Equilibration buffer (LEW, 50 mM NaH ₂ PO ₄ , 300 mM NaCl, 1 mM Immidazole, pH8.0), and lysozyme (MERCK) was added to the total concentration at 1 mg / ml. This was added to incubate on ice for 30 minutes. Thereafter, the cells were ground by sonication (Ultrasonication) and only the pellet portion was left through centrifugation (13,000 rpm, 30 minutes) of cell-lysate. The pellet was then lysed with Lysis-Equilibration buffer and centrifuged (13,000 rpm, 30 minutes) to wash the pellet. Lysis buffer (8M Urea, 50mM NaH ₂ PO ₄ , 300mM NaCl, 5mM Immidazole, pH8.0) was added to the pellet to denature the protein. After sonication of the pellet to be well suspended and incubated for 30 minutes at room temperature, water-soluble proteins were prepared by centrifugation (13,000 rpm, 30 minutes) of cell-lysate, and an injection filter of 0.45 μm. filter, Sartorius).

In order to elute the fusion peptide, FPLC (Bio-Rad, BioLogic DuoFlow Standard System) equipment was used. Two types of buffers (buffer A: 8M Urea, 50mM NaH ₂ PO ₄ , 300mM NaCl, 5mM Immidazole, pH8) using Ni-NTA His bind resin (Bio-Rad), one of the affinity chromatography methods .0, buffer B: 8M Urea, 50 mM NaH ₂ PO ₄ , 300 mM NaCl, 250 mM Immidazole, pH 8.0) was eluted with the fusion peptide using the gradient method. (FIGS. 10A and 11A).

The eluted fragments were analyzed on 15% SDS-PAGE. Purified fusion peptide was selected to slowly remove Urea through buffer substitution method for refolding. Substitute the buffer overnight in the final 2 mM borate (Sodium Tetraborate, pH9.0), Refrigerated through.

Purified electrophoresis pictures of the fusion peptides of the recombinant ERNS-F2 and ERNS-F3 antigen proteins are shown in FIGS. 10B and 11B.

Example  2. Recombination ERNS  Screening of -F2 and -F3 Antigen Proteins

A. Recombination ERNS Of -F2 and -F3 antigenic proteins dot - immunoassay Screening via

For antigenicity analysis of the ERNS-F2 and -F3 recombinant antigens, the antigens that specifically reacted with the antigens purified in Example 1D were reselected by Dot Immunoassay and used as diagnostic antigens ( 1).

Dot immunoassay was performed according to the following procedure.

Recombinant ERNS-F2 and -F3 antigenic proteins were adsorbed onto nitrocellulose membranes using 96-well hybrid-dot manifold (GibcoBRL, Gaitherburg, Mo.). Thereafter, blocking was performed for 1 hour at room temperature with a blocking solution (5% nonfatmilk-PBS solution) to inhibit the nonspecific reaction. Swine fever virus positive sample diluted in 1/50 ratio in 2% BSA-PBS solution was reacted by shaking slowly for 2 hours at room temperature. The membrane to which the sample reacted was washed in the membrane three times for 10 minutes with TBST buffer (20 mM Tris pH 8.0, 137 mM NaCl, 0.05% Tween-20) or PBST (1X PBS, 0.05% Tween-20). After removing the substance, HRP (houseradish peroxidase) -conjugated anti-swine IgG secondary antibody (diluted at 1/2000 magnification with 5% nonfatmilk-TBST) (Santa Cruze) was reacted with shaking at room temperature for 1 hour, followed by TBST buffer. The membrane was washed five times for 10 minutes. Finally, the membrane was reacted with Chemiluminescence Luminol reagent (GE Heathcare) for 1 minute at room temperature and then exposed to X-ray film (FUJIFILM) for 30 seconds to 4 minutes (Fig. 1).

B. Recombination ERNS Direct enzyme immunoassay of -F2 and -F3 antigenic proteins direct  Screening via ELISA

For antigenicity analysis of the ERNS-F2 and -F3 antigen proteins, the antigens purified in Example 1D were each concentrated in coating buffer (KOMA BIOTECH INC, K0331021) at 0, 100, 250, 500, 1000 (ng / ml) concentration. Diluted to 100 ㎕ per well and coated with overnight incubation at 4 ℃. The coated plate was washed three times using PBS, and blocking was performed by adding Blocking solution (1% BSA / PBS) by 250 μl per well for 1 hour at room temperature. The blocked plate was washed again 5 times using PBS washing solution, and 200 μl of the swine fever virus antibody positive sample diluted 1:50 was added thereto and reacted at room temperature for 1 hour. After the sample reaction was completed, the plate was washed three times using PBS washing solution, and 100 μl of anti-swine IgG HRP (1: 5,000, Antibody Incorporated, USA) was added per well for 1 hour at room temperature. After the process, the color reaction was induced with TMB Substrate kit (KOMA BIOTECH INC, K0331060) and the reaction was stopped with stop solution (2 M, H ₂ SO ₄ ) and analyzed by ELISA reader (TECAN) 450nm wavelength absorbance. (FIG. 2).

C. Results

As a result of Examples 2A and 2B, it was confirmed that the two recombinant antigens of Example 1 of the present invention responded to Swine fever virus infection-positive specimens 1 to 3 (Specimen 1-3) but their antigenicity depending on the characteristics of the specimen. This differentiation was confirmed (FIGS. 1 and 2). In addition, the ERNS-F2 fusion peptide of the two recombinant antigens in particular, the reactivity was higher than the ERNS-F3 fusion peptide in all the samples it was confirmed that it has a relatively high antigenicity.

Thus, the efficacy of the recombinant antigen comprising the sequence of SEQ ID NO: 4 and the recombinant antigen comprising the sequence of SEQ ID NO: 6 has been demonstrated, and the recombinant antigen exhibiting such reactivity was used in the production of a diagnostic kit according to the present invention.

Example  3. Swine Fever Diagnosis System  Diagnostic Strips and Diagnostics Of kit  Produce

A. Swine Fever Virus ERNS  Antigen-coated Of the membrane  Ready

The recombinant proteins ERNS-F2 (SEQ ID NO: 4) and ERNS-F3 (SEQ ID NO: 6), stored in 2 mM sodium tetraborate, were used as test lines at a concentration of 2 mg / ml, and the control line was goat anti-mouse immunoglobulin. Goat anti-mouse IgG (G) (Santa Cruze) was used. Recombinant antigens of the test line and control solution of the control line were coated onto the nitro cellulose membrane using a dispensing instrument (KINAMETICS, USA). It was then dried overnight in a low humidity laboratory or in a pan for at least 5 hours. The plate of membrane prepared above was stored in a container sealed with desiccant or in a low humidity laboratory.

B. Swine Fever Virus ERNS  Antigen gold ( Gold ) Manufacture of conjugate

The swine fever virus ERNS-F2 / -F3 antigen or protein A (Sigma) was added dropwise to the gold solution with stirring to a final concentration of 200 μg / ml, and then the solution was stirred for another 15 minutes. Thereafter, 10% BSA solution was added to the gold particle suspension. After another 15 minutes of stirring, the bound gold solution was centrifuged to discard the supernatant to remove unbound recombinant protein, and the combined gold solution pellets were 0.1% Casein and 1% BSA three times the pellet volume. The pellet was resuspended after the addition of 5 mM sodium borate (pH 7.2). The suspension was centrifuged again, and the final pellet was adjusted to 5 ± 1 mM Sodium Tetraborate (pH 7.2) with 0.1% of Casein and 1% of BSA so that the absorbance was 10 ± 1 at 530 nm of spectrophotometer. The swine fever virus ERNS antigen Gold conjugate was prepared.

C. Mouse Immunoglobulin  G-gold conjugate (( Mouse IgG - Gold Conjugate ) Preparation: Control Indicator of Antibody Diagnostic System

To prepare gold conjugates for the control line of the antibody diagnostic system, mouse IgG purchased from Arista, USA was added dropwise to the gold solution with stirring until the final concentration was 4-20 μg / ml. The solution was stirred again for 15 minutes. Thereafter, 10% BSA solution (Promega) was added to the gold particle suspension. After another 15 minutes of stirring, the bound gold solution was centrifuged to discard the supernatant in order to remove unbound recombinant protein, and 5 mM of 1% BSA added to the bound gold solution pellet was added to the pellet. The pellet was resuspended after the addition of borate (Sodium Tetraborate, pH7.2). The suspension was centrifuged again and the final pellet was prepared by adjusting the absorbance to 10 +/- 1 at 530 nm of spectrophotometer in 5 mM borate (Sodium Tetraborate, pH 7.2) with 1% BSA.

D. Preparation of gold conjugate treatment pad

Gold conjugates prepared in Examples 3B and 3C were prepared as follows by the addition of 5% Trehalose.

For swine fever virus ERNS antibody assay strips, the swine fever virus ERNS antigen gold conjugate prepared in Example 3B or protein A gold conjugate was prepared in 0.5 x 20 cm glass fiber at a final concentration of 0.5 to 2.5 OD (Optical Density). And to the mouse IgG-gold conjugate prepared in Example 3C to a final concentration of 0.5 OD.

E. Absorption Pads and Sample pad  Produce

Absorbent pad and sample pad were prepared by drying so that the reaction solution can be absorbed well.

F. device  Assembly

By selecting one of the diagnostic products prepared above, each of the membranes and pads, the sample pad, the gold pad (gold conjugate pad), the nitrocellulose membrane, and finally the absorbent pad are combined to overlap each other from the right side, and immunoassay device (Device) was cut into strip sizes that matched the size. The strip thus cut was finally put in the lower plate of the single immunoassay device (single device) and the top plate was covered to prepare a diagnostic kit (FIG. 3).

G. Product Composition

The immunoassay device and sample development (PBS, 0.5% BSA, 0.1% NaN 3) described above were configured to be the final product (FIG. 3).

<110> VETALL CO., LTD. <120> ANTIGEN FOR DETECTING OF CLASSICAL SWINE FEVER VIRUS, ANTIBODY          DETECTING METHOD AND TEST KIT USING THEREOF <130> DPP20110316KR <160> 22 <170> Kopatentin 1.71 <210> 1 <211> 3898 <212> PRT <213> Artificial Sequence <220> <223> Classical swine fever virus <400> 1 Met Glu Leu Asn His Phe Glu Leu Leu Tyr Lys Thr Asn Glu Gln Lys   1 5 10 15 Pro Met Gly Val Glu Glu Pro Val Tyr Asp Ala Thr Gly Lys Pro Leu              20 25 30 Phe Gly Asp Pro Ser Glu Val His Pro Gln Ser Thr Leu Lys Leu Pro          35 40 45 His Asp Arg Gly Arg Gly Asn Ile Lys Thr Thr Leu Lys Asn Leu Pro      50 55 60 Arg Lys Gly Asp Cys Arg Ser Gly Asn His Leu Gly Pro Val Ser Gly  65 70 75 80 Ile Tyr Val Lys Pro Gly Pro Val Phe Tyr Gln Asp Tyr Met Gly Pro                  85 90 95 Val Tyr His Arg Ala Pro Leu Glu Phe Phe Ser Glu Ala Gln Phe Cys             100 105 110 Glu Val Thr Lys Arg Ile Gly Arg Val Thr Gly Ser Asp Gly Arg Leu         115 120 125 Tyr His Ile Tyr Val Cys Ile Asp Gly Cys Ile Leu Leu Lys Leu Ala     130 135 140 Lys Arg Gly Glu Pro Arg Thr Leu Lys Trp Ile Arg Asn Phe Thr Asp 145 150 155 160 Cys Pro Leu Trp Val Thr Ser Cys Ser Asp Asp Gly Ala Ser Gly Ser                 165 170 175 Lys Glu Lys Lys Pro Asp Arg Ile Asn Lys Gly Lys Leu Lys Ile Ala             180 185 190 Pro Lys Glu His Glu Lys Asp Ser Arg Thr Lys Pro Pro Asp Ala Thr         195 200 205 Ile Val Val Glu Gly Val Lys Tyr Gln Val Arg Lys Lys Gly Lys Val     210 215 220 Lys Gly Lys Asn Thr Gln Asp Gly Leu Tyr His Asn Lys Asn Lys Pro 225 230 235 240 Pro Glu Ser Arg Lys Lys Leu Glu Lys Thr Leu Leu Ala Trp Ala Val                 245 250 255 Ile Ala Ile Met Leu Tyr Gln Pro Val Glu Ala Glu Asn Ile Thr Gln             260 265 270 Trp Asn Leu Ser Asp Asn Gly Thr Asn Gly Ile Gln His Ala Met Tyr         275 280 285 Leu Arg Gly Ile Ser Arg Ser Leu His Gly Ile Trp Pro Glu Lys Ile     290 295 300 Cys Lys Gly Val Pro Thr Tyr Leu Ala Thr Asp Thr Glu Leu Lys Glu 305 310 315 320 Ile Gln Gly Met Met Asp Ala Ser Glu Gly Thr Asn Tyr Thr Cys Cys                 325 330 335 Lys Leu Gln Arg His Glu Trp Asn Lys His Gly Trp Cys Asn Trp Tyr             340 345 350 Asn Ile Asp Pro Trp Ile Gln Leu Met Asn Arg Thr Gln Ala Asn Leu         355 360 365 Ala Glu Gly Pro Pro Ala Lys Glu Cys Ala Val Thr Cys Arg Tyr Asp     370 375 380 Lys Asp Ala Asp Val Asn Val Val Thr Gln Ala Arg Asn Arg Pro Thr 385 390 395 400 Thr Leu Thr Gly Cys Lys Lys Gly Lys Asn Phe Ser Phe Ala Gly Thr                 405 410 415 Val Ile Glu Gly Pro Cys Asn Phe Asn Val Ser Val Glu Asp Ile Leu             420 425 430 Tyr Gly Asp His Glu Cys Gly Ser Leu Leu Gln Asp Thr Ala Leu Tyr         435 440 445 Leu Val Asp Gly Met Thr Asn Thr Ile Glu Asn Ala Arg Gln Gly Ala     450 455 460 Ala Arg Val Thr Ser Trp Leu Gly Arg Gln Leu Arg Ile Ala Gly Arg 465 470 475 480 Arg Leu Glu Gly Arg Ser Lys Thr Trp Phe Gly Ala Tyr Ala Leu Ser                 485 490 495 Pro Tyr Cys Asn Val Thr Ser Lys Ile Gly Tyr Ile Trp Tyr Thr Asn             500 505 510 Asn Cys Thr Pro Ala Cys Leu Pro Lys Asn Thr Lys Ile Ily Gly Pro         515 520 525 Gly Lys Phe Asp Thr Asn Ala Glu Asp Gly Lys Ile Leu His Glu Met     530 535 540 Gly Gly His Leu Ser Glu Phe Leu Leu Leu Ser Leu Val Val Leu Ser 545 550 555 560 Asp Phe Ala Pro Glu Thr Ala Ser Ala Leu Tyr Leu Ile Phe His Tyr                 565 570 575 Val Ile Pro Gln Ser His Glu Glu Leu Glu Gly Cys Asp Thr Asn Gln             580 585 590 Leu Asn Leu Thr Val Glu Leu Arg Thr Glu Asp Val Ile Pro Ser Ser         595 600 605 Val Trp Asn Val Gly Lys Tyr Val Cys Val Arg Pro Asp Trp Trp Pro     610 615 620 Tyr Glu Thr Lys Val Ala Leu Leu Phe Glu Glu Ala Gly Gln Val Val 625 630 635 640 Lys Leu Ala Val Arg Ala Leu Arg Asp Leu Thr Arg Val Trp Asn Ser                 645 650 655 Ala Ser Thr Thr Ala Phe Leu Ile Cys Leu Ile Lys Val Leu Arg Gly             660 665 670 Gln Ile Val Gln Gly Val Ile Trp Leu Leu Leu Val Thr Gly Ala Gln         675 680 685 Gly Gln Leu Ala Cys Lys Glu Asp Tyr Arg Tyr Ala Ile Ser Ser Thr     690 695 700 Asn Glu Ile Gly Leu Leu Gly Ala Gly Gly Leu Thr Thr Thr Trp Lys 705 710 715 720 Glu Tyr Asn His Asp Leu Gln Leu Asn Asp Gly Thr Val Lys Ala Ile                 725 730 735 Cys Val Ala Gly Ser Phe Lys Val Thr Ala Leu Asn Val Val Ser Arg             740 745 750 Arg Tyr Leu Ala Ser Leu His Lys Glu Ala Leu Pro Thr Ser Val Ala         755 760 765 Phe Glu Leu Leu Phe Asp Gly Thr Asn Pro Ser Thr Glu Glu Met Gly     770 775 780 Asp Asp Phe Gly Phe Gly Gln Cys Pro Phe Asp Thr Ser Pro Val Val 785 790 795 800 Lys Gly Lys Tyr Asn Thr Thr Leu Leu Asn Gly Ser Ala Phe Tyr Leu                 805 810 815 Val Cys Pro Ile Gly Trp Thr Gly Val Ile Glu Cys Thr Ala Val Ser             820 825 830 Pro Thr Thr Leu Arg Thr Glu Val Val Lys Thr Phe Arg Arg Asp Lys         835 840 845 Pro Phe Pro His Arg Met Phe Cys Val Thr Thr Thr Thr Val Glu Asn Glu     850 855 860 Asp Leu Phe Tyr Cys Lys Leu Gly Gly Asn Trp Thr Cys Val Lys Gly 865 870 875 880 Glu Pro Val Val Tyr Thr Gly Gly Leu Val Lys Gln Cys Arg Trp Cys                 885 890 895 Gly Phe Asp Phe Asn Glu Pro Asp Gly Leu Pro His Tyr Pro Ile Gly             900 905 910 Lys Cys Ile Leu Ala Asn Glu Thr Gly Tyr Arg Ile Val Asp Ser Thr         915 920 925 Asp Cys Asn Arg Asp Gly Val Val Ile Ser Thr Glu Gly Ser His Glu     930 935 940 Cys Leu Ile Gly Asn Thr Thr Val Lys Val His Ala Ser Asp Glu Arg 945 950 955 960 Leu Gly Pro Met Pro Cys Arg Pro Lys Glu Ile Val Ser Ser Ala Gly                 965 970 975 Pro Val Arg Lys Thr Ser Cys Thr Phe Asn Tyr Ala Lys Thr Leu Lys             980 985 990 Asn Lys Tyr Tyr Glu Pro Arg Asp Ser Tyr Phe Gln Gln Tyr Met Leu         995 1000 1005 Lys Gly Glu Tyr Gln Tyr Trp Phe Asp Leu Asp Val Thr Asp Arg His    1010 1015 1020 Ser Asp Tyr Phe Ala Glu Phe Val Val Leu Val Val Val Ala Leu Leu 1025 1030 1035 1040 Gly Gly Arg Tyr Ile Leu Trp Leu Ile Val Thr Tyr Ile Val Leu Thr                1045 1050 1055 Glu Gln Pro Ala Ala Gly Leu Pro Leu Gly Gln Gly Glu Val Val Leu            1060 1065 1070 Ile Gly Asn Leu Ile Thr His Thr Asp Ile Glu Val Val Val Tyr Phe        1075 1080 1085 Leu Leu Leu Tyr Leu Val Met Arg Asp Glu Pro Ile Lys Lys Trp Ile    1090 1095 1100 Leu Leu Leu Phe His Ala Ile Thr Asn Asn Pro Val Lys Thr Ile Thr 1105 1110 1115 1120 Val Ala Leu Leu Met Val Ser Gly Val Ala Arg Gly Gly Lys Ile Asp                1125 1130 1135 Gly Gly Trp Gln Arg Leu Pro Glu Thr Ser Phe Asp Ile Gln Leu Ala            1140 1145 1150 Leu Thr Val Ile Val Val Ala Val Met Leu Leu Ala Lys Arg Asp Pro        1155 1160 1165 Thr Thr Val Pro Leu Val Val Thr Val Ala Thr Leu Arg Thr Ala Lys    1170 1175 1180 Met Thr Asn Gly Leu Ser Thr Asp Ile Ala Ile Ala Thr Val Ser Thr 1185 1190 1195 1200 Ala Leu Leu Thr Trp Thr Tyr Ile Ser Asp Tyr Tyr Arg Tyr Lys Thr                1205 1210 1215 Trp Leu Gln Tyr Leu Ile Ser Thr Val Thr Gly Ile Phe Leu Ile Arg            1220 1225 1230 Val Leu Lys Gly Ile Gly Glu Leu Asp Leu His Thr Pro Thr Leu Pro        1235 1240 1245 Ser Tyr Arg Pro Leu Phe Phe Ile Leu Val Tyr Leu Ile Ser Thr Ala    1250 1255 1260 Val Val Thr Arg Trp Asn Leu Asp Ile Ala Gly Leu Leu Leu Gln Cys 1265 1270 1275 1280 Ala Pro Thr Leu Leu Met Val Phe Thr Met Trp Ala Asp Ile Leu Thr                1285 1290 1295 Leu Ile Leu Ile Leu Pro Thr Tyr Glu Leu Thr Lys Leu Tyr Tyr Leu            1300 1305 1310 Lys Glu Val Lys Ile Gly Ala Glu Arg Gly Trp Leu Trp Lys Thr Asn        1315 1320 1325 Phe Lys Arg Val Asn Asp Ile Tyr Glu Val Asp Gln Ala Gly Glu Gly    1330 1335 1340 Val Tyr Leu Phe Pro Ser Lys Gln Lys Thr Ser Ser Ile Thr Gly Thr 1345 1350 1355 1360 Met Leu Pro Leu Ile Lys Ala Ile Leu Ile Ser Cys Ile Ser Asn Lys                1365 1370 1375 Trp Gln Phe Ile Tyr Leu Leu Tyr Leu Ile Phe Glu Val Ser Tyr Tyr            1380 1385 1390 Leu His Lys Lys Ile Ile Asp Glu Ile Ala Gly Gly Thr Asn Phe Ile        1395 1400 1405 Ser Arg Leu Val Ala Ala Leu Ile Glu Ala Asn Trp Ala Phe Asp Asn    1410 1415 1420 Glu Glu Val Arg Gly Leu Lys Lys Phe Phe Leu Leu Ser Ser Arg Val 1425 1430 1435 1440 Lys Glu Leu Ile Ile Lys His Lys Val Arg Asn Glu Val Met Val His                1445 1450 1455 Trp Phe Gly Asp Glu Glu Val Tyr Gly Met Pro Lys Leu Val Gly Leu            1460 1465 1470 Val Lys Ala Ala Thr Leu Ser Lys Asn Lys His Cys Ile Leu Cys Thr        1475 1480 1485 Val Cys Glu Asp Arg Glu Trp Arg Gly Glu Thr Cys Pro Lys Cys Gly    1490 1495 1500 Arg Phe Gly Pro Pro Met Thr Cys Gly Met Thr Leu Ala Asp Phe Glu 1505 1510 1515 1520 Glu Lys His Tyr Lys Arg Ile Phe Phe Arg Glu Asp Gln Ser Glu Gly                1525 1530 1535 Pro Val Arg Glu Glu Tyr Ala Gly Tyr Leu Gln Tyr Arg Ala Arg Gly            1540 1545 1550 Gln Leu Phe Leu Arg Asn Leu Pro Val Leu Ala Thr Lys Val Lys Met        1555 1560 1565 Leu Leu Val Gly Asn Leu Gly Thr Glu Val Gly Asp Leu Glu His Leu    1570 1575 1580 Gly Trp Val Leu Arg Gly Pro Ala Val Cys Lys Lys Val Thr Glu His 1585 1590 1595 1600 Glu Lys Cys Thr Thr Ser Ile Met Asp Lys Leu Thr Ala Phe Phe Gly                1605 1610 1615 Val Met Pro Arg Gly Thr Thr Pro Arg Ala Pro Val Arg Phe Pro Thr            1620 1625 1630 Ser Leu Leu Lys Ile Arg Arg Gly Leu Glu Thr Gly Trp Ala Tyr Thr        1635 1640 1645 His Gln Gly Gly Ile Ser Ser Val Asp His Val Thr Cys Gly Lys Asp    1650 1655 1660 Leu Leu Val Cys Asp Thr Met Gly Arg Thr Arg Val Val Cys Gln Ser 1665 1670 1675 1680 Asn Asn Lys Met Thr Asp Glu Ser Glu Tyr Gly Val Lys Thr Asp Ser                1685 1690 1695 Gly Cys Pro Glu Gly Ala Arg Cys Tyr Val Phe Asn Pro Glu Ala Val            1700 1705 1710 Asn Ile Ser Gly Thr Lys Gly Ala Met Val His Leu Gln Lys Thr Gly        1715 1720 1725 Gly Glu Phe Thr Cys Val Thr Ala Ser Gly Thr Pro Ala Phe Phe Asp    1730 1735 1740 Leu Lys Asn Leu Lys Gly Trp Ser Gly Leu Pro Ile Phe Glu Ala Ser 1745 1750 1755 1760 Ser Gly Arg Val Val Gly Arg Val Lys Val Gly Lys Asn Glu Asp Ser                1765 1770 1775 Lys Pro Thr Lys Leu Met Ser Gly Ile Gln Thr Val Ser Lys Ser Thr            1780 1785 1790 Thr Asp Leu Thr Glu Met Val Lys Lys Ile Thr Thr Met Asn Arg Gly        1795 1800 1805 Glu Phe Arg Gln Ile Thr Leu Ala Thr Gly Ala Gly Lys Thr Thr Glu    1810 1815 1820 Leu Pro Arg Ser Val Ile Glu Glu Ile Gly Arg His Lys Arg Val Leu 1825 1830 1835 1840 Val Leu Ile Pro Leu Arg Ala Ala Ala Glu Ser Val Tyr Gln Tyr Met                1845 1850 1855 Arg Gln Lys His Pro Ser Ile Ala Phe Asn Leu Arg Ile Gly Glu Met            1860 1865 1870 Lys Glu Gly Asp Met Ala Thr Gly Ile Thr Tyr Ala Ser Tyr Gly Tyr        1875 1880 1885 Phe Cys Gln Met Pro Gln Pro Lys Leu Arg Ala Ala Met Val Glu Tyr    1890 1895 1900 Ser Phe Ile Phe Leu Asp Glu Tyr His Cys Ala Thr Pro Glu Gln Leu 1905 1910 1915 1920 Ala Ile Met Gly Lys Ile His Arg Phe Ser Glu Asn Leu Arg Val Val                1925 1930 1935 Ala Met Thr Ala Thr Pro Ala Gly Thr Val Thr Thr Thr Gly Gln Lys            1940 1945 1950 His Pro Ile Glu Glu Phe Ile Ala Pro Glu Val Met Lys Gly Glu Asp        1955 1960 1965 Leu Gly Ser Glu Tyr Leu Asp Ile Ala Gly Leu Lys Ile Pro Val Glu    1970 1975 1980 Glu Met Lys Ser Asn Met Leu Val Phe Val Pro Thr Arg Asn Met Ala 1985 1990 1995 2000 Val Glu Thr Ala Lys Lys Leu Lys Ala Lys Gly Tyr Asn Ser Gly Tyr                2005 2010 2015 Tyr Tyr Ser Gly Glu Asp Pro Ser Asn Leu Arg Val Val Thr Ser Gln            2020 2025 2030 Ser Pro Tyr Val Val Val Ala Thr Asn Ala Ile Glu Ser Gly Val Thr        2035 2040 2045 Leu Pro Asp Leu Asp Val Val Val Asp Thr Gly Leu Lys Cys Glu Lys    2050 2055 2060 Arg Ile Arg Leu Ser Pro Lys Met Pro Phe Ile Val Thr Gly Leu Lys 2065 2070 2075 2080 Arg Met Ala Val Thr Ile Gly Glu Gln Ala Gln Arg Arg Gly Arg Val                2085 2090 2095 Gly Arg Val Lys Pro Gly Arg Tyr Tyr Arg Ser Gln Glu Thr Pro Val            2100 2105 2110 Gly Ser Lys Asp Tyr His Tyr Asp Leu Leu Gln Ala Gln Arg Tyr Gly        2115 2120 2125 Ile Glu Asp Gly Ile Asn Ile Thr Lys Ser Phe Arg Glu Met Asn Tyr    2130 2135 2140 Asp Trp Ser Leu Tyr Glu Glu Asp Ser Leu Met Ile Thr Gln Leu Glu 2145 2150 2155 2160 Ile Leu Asn Asn Leu Leu Ile Ser Glu Glu Leu Pro Met Ala Val Lys                2165 2170 2175 Asn Ile Met Ala Arg Thr Asp His Pro Glu Pro Ile Gln Leu Ala Tyr            2180 2185 2190 Asn Ser Tyr Glu Thr Gln Val Pro Val Leu Phe Pro Lys Ile Lys Asn        2195 2200 2205 Gly Glu Val Thr Asp Ser Tyr Asp Asn Tyr Thr Phe Leu Asn Ala Arg    2210 2215 2220 Lys Leu Gly Asp Asp Val Pro Pro Tyr Val Tyr Ala Thr Glu Asp Glu 2225 2230 2235 2240 Asp Leu Ala Val Glu Leu Leu Gly Leu Asp Trp Pro Asp Pro Gly Asn                2245 2250 2255 Gln Gly Thr Val Glu Val Gly Arg Ala Leu Lys Gln Val Val Gly Leu            2260 2265 2270 Ser Thr Ala Glu Asn Ala Leu Leu Val Ala Leu Phe Gly Tyr Val Gly        2275 2280 2285 Tyr Gln Ala Leu Ser Lys Arg His Ile Pro Val Val Thr Asp Ile Tyr    2290 2295 2300 Ser Ile Glu Asp His Arg Leu Glu Asp Thr Thr His Leu Gln Tyr Ala 2305 2310 2315 2320 Pro Asn Ala Ile Lys Thr Glu Gly Lys Glu Thr Glu Leu Lys Glu Leu                2325 2330 2335 Ala Gln Gly Asp Val Gln Arg Cys Val Gly Ala Met Thr Asn Tyr Ala            2340 2345 2350 Arg Glu Gly Ile Gln Phe Met Lys Ser Gln Ala Leu Lys Val Lys Glu        2355 2360 2365 Thr Pro Thr Tyr Lys Glu Thr Met Asp Thr Val Thr Asp Tyr Val Lys    2370 2375 2380 Lys Phe Met Glu Ala Leu Thr Asp Ser Lys Glu Asp Ile Ile Lys Tyr 2385 2390 2395 2400 Gly Leu Trp Gly Thr His Thr Ala Leu Tyr Lys Ser Ile Cys Ala Arg                2405 2410 2415 Leu Gly Ser Glu Thr Ala Phe Ala Thr Leu Val Val Lys Trp Leu Ala            2420 2425 2430 Phe Gly Gly Glu Ser Ile Ala Asp His Val Lys Gln Ala Ala Thr Asp        2435 2440 2445 Leu Val Val Tyr Tyr Ile Ile Asn Arg Pro Gln Phe Pro Gly Asp Thr    2450 2455 2460 Glu Thr Gln Gln Glu Gly Arg Lys Tyr Val Ala Ser Leu Leu Val Ser 2465 2470 2475 2480 Ala Leu Val Thr Tyr Thr Tyr Lys Ser Trp Asn Tyr Asn Asn Leu Ser                2485 2490 2495 Lys Ile Val Glu Pro Ala Leu Ala Thr Leu Pro Tyr Ala Ala Thr Ala            2500 2505 2510 Leu Lys Leu Phe Ala Pro Thr Arg Leu Glu Ser Val Val Ile Leu Ser        2515 2520 2525 Thr Ala Ile Tyr Lys Thr Tyr Leu Ser Ile Arg Arg Gly Lys Ser Asp    2530 2535 2540 Gly Leu Leu Gly Thr Gly Val Ser Ala Ala Met Glu Ile Met Ser Gln 2545 2550 2555 2560 Asn Pro Val Ser Val Gly Ile Ala Val Met Leu Gly Val Gly Ala Val                2565 2570 2575 Ala Ala His Asn Ala Ile Glu Ala Ser Glu Gln Lys Arg Thr Leu Leu            2580 2585 2590 Met Lys Val Phe Val Lys Asn Phe Leu Asp Gln Ala Ala Thr Asp Glu        2595 2600 2605 Leu Val Lys Glu Ser Pro Glu Lys Ile Ile Met Ala Leu Phe Glu Ala    2610 2615 2620 Val Gln Thr Val Gly Asn Pro Leu Arg Leu Val Tyr His Leu Tyr Gly 2625 2630 2635 2640 Val Phe Tyr Lys Gly Trp Glu Ala Lys Glu Leu Ala Gln Arg Thr Ala                2645 2650 2655 Gly Arg Asn Leu Phe Thr Leu Ile Met Phe Glu Ala Val Glu Leu Leu            2660 2665 2670 Gly Val Asp Ser Glu Gly Lys Ile Arg Gln Leu Ser Ser Asn Tyr Ile        2675 2680 2685 Leu Glu Leu Leu Tyr Lys Phe Arg Asp Ser Ile Lys Ser Ser Val Arg    2690 2695 2700 Asp Met Ala Ile Ser Trp Ala Pro Ala Pro Phe Ser Cys Asp Trp Thr 2705 2710 2715 2720 Pro Thr Asp Asp Arg Ile Gly Leu Pro Gln Asp Asn Phe Leu Gln Val                2725 2730 2735 Glu Thr Lys Cys Pro Cys Gly Tyr Lys Met Lys Ala Val Lys Asn Cys            2740 2745 2750 Ala Gly Glu Leu Arg Leu Leu Glu Glu Glu Gly Ser Phe Leu Cys Arg        2755 2760 2765 Asn Lys Phe Gly Arg Gly Ser Arg Asn Tyr Arg Val Thr Lys Tyr Tyr    2770 2775 2780 Asp Asp Asn Leu Ser Glu Ile Lys Pro Val Ile Arg Met Glu Gly His 2785 2790 2795 2800 Val Glu Leu Tyr Tyr Lys Gly Ala Thr Ile Lys Leu Asp Phe Asn Asn                2805 2810 2815 Ser Lys Thr Ile Leu Ala Thr Asp Lys Trp Glu Val Asp His Ser Thr            2820 2825 2830 Leu Val Arg Val Leu Lys Arg His Thr Gly Ala Gly Tyr His Gly Ala        2835 2840 2845 Tyr Leu Gly Glu Lys Pro Asn His Lys His Leu Ile Glu Arg Asp Cys    2850 2855 2860 Ala Thr Ile Thr Lys Asp Lys Val Cys Phe Leu Lys Met Lys Arg Gly 2865 2870 2875 2880 Cys Ala Phe Thr Tyr Asp Leu Ser Leu His Asn Leu Thr Arg Leu Ile                2885 2890 2895 Glu Leu Val His Lys Asn Asn Leu Glu Asp Lys Glu Ile Pro Ala Val            2900 2905 2910 Thr Val Thr Thr Trp Leu Ala Tyr Thr Phe Val Asn Glu Asp Ile Gly        2915 2920 2925 Thr Ile Lys Pro Ala Phe Gly Glu Lys Val Thr Pro Glu Met Gln Glu    2930 2935 2940 Glu Ile Thr Leu Gln Pro Ala Val Val Val Asp Thr Thr Asp Val Thr 2945 2950 2955 2960 Val Thr Val Val Gly Glu Ala Pro Thr Met Thr Thr Gly Glu Thr Pro                2965 2970 2975 Thr Ala Phe Thr Ser Ser Gly Ser Asp Pro Lys Gly Gln Gln Val Leu            2980 2985 2990 Lys Leu Gly Val Gly Glu Gly Gln Tyr Pro Gly Thr Asn Pro Gln Arg        2995 3000 3005 Ala Ser Leu His Glu Ala Ile Gln Gly Ala Asp Glu Arg Pro Ser Val    3010 3015 3020 Leu Ile Leu Gly Ser Asp Lys Ala Thr Ser Asn Arg Val Lys Thr Ala 3025 3030 3035 3040 Lys Asn Val Lys Val Tyr Arg Gly Arg Asp Pro Leu Glu Val Arg Asp                3045 3050 3055 Met Met Arg Arg Gly Lys Ile Leu Val Ile Ala Leu Ser Arg Val Asp            3060 3065 3070 Asn Ala Leu Leu Lys Phe Val Asp Tyr Lys Gly Thr Phe Leu Thr Arg        3075 3080 3085 Glu Thr Leu Glu Ala Leu Ser Leu Gly Arg Pro Lys Lys Lys Asn Ile    3090 3095 3100 Thr Lys Ala Glu Ala Gln Trp Leu Leu Cys Leu Glu Asp Gln Met Glu 3105 3110 3115 3120 Glu Leu Pro Asp Trp Phe Ala Ala Gly Glu Pro Ile Phe Leu Glu Ala                3125 3130 3135 Asn Ile Lys His Asp Arg Tyr His Leu Val Gly Asp Ile Ala Thr Ile            3140 3145 3150 Lys Glu Lys Ala Lys Gln Leu Gly Ala Thr Asp Ser Thr Lys Ile Ser        3155 3160 3165 Lys Glu Val Gly Ala Lys Val Tyr Ser Met Lys Leu Ser Asn Trp Val    3170 3175 3180 Met Gln Glu Glu Asn Lys Gln Gly Asn Leu Thr Pro Leu Phe Glu Glu 3185 3190 3195 3200 Leu Leu Gln Gln Cys Pro Pro Gly Gly Gln Asn Lys Thr Ala His Met                3205 3210 3215 Val Ser Ala Tyr Gln Leu Ala Gln Gly Asn Trp Met Pro Thr Ser Cys            3220 3225 3230 His Val Phe Met Gly Thr Ile Ser Ala Arg Arg Thr Lys Thr His Pro        3235 3240 3245 Tyr Glu Ala Tyr Val Lys Leu Arg Glu Leu Val Glu Glu His Lys Met    3250 3255 3260 Lys Thr Leu Cys Pro Gly Ser Ser Leu Gly Lys His Asn Glu Trp Ile 3265 3270 3275 3280 Ile Gly Lys Ile Lys Tyr Gln Gly Asn Leu Arg Thr Lys His Met Leu                3285 3290 3295 Asn Pro Gly Lys Val Ala Glu Gln Leu Cys Arg Glu Gly His Arg Arg            3300 3305 3310 Asn Val Tyr Asn Lys Thr Ile Gly Ser Val Met Thr Ala Thr Gly Ile        3315 3320 3325 Arg Leu Glu Lys Leu Pro Val Val Arg Ala Gln Thr Asp Thr Thr Asn    3330 3335 3340 Phe His Gln Ala Ile Arg Asp Lys Ile Asp Lys Glu Glu Asn Leu Gln 3345 3350 3355 3360 Thr Pro Gly Leu His Lys Lys Leu Met Glu Val Phe Asn Ala Leu Lys                3365 3370 3375 Arg Pro Glu Leu Glu Ser Ser Tyr Asp Ala Val Glu Trp Glu Glu Leu            3380 3385 3390 Glu Arg Gly Ile Asn Arg Lys Gly Ala Ala Gly Phe Phe Glu Arg Lys        3395 3400 3405 Asn Ile Gly Glu Ile Leu Asp Ser Glu Lys Asn Lys Val Glu Glu Ile    3410 3415 3420 Ile Asp Asn Leu Lys Lys Gly Arg Asn Ile Lys Tyr Tyr Glu Thr Ala 3425 3430 3435 3440 Ile Pro Lys Asn Glu Lys Arg Asp Val Asn Asp Asp Trp Thr Ser Gly                3445 3450 3455 Asp Phe Val Asp Glu Lys Lys Pro Arg Val Ile Gln Tyr Pro Glu Ala            3460 3465 3470 Lys Thr Arg Leu Ala Ile Thr Lys Val Met Tyr Lys Trp Val Lys Gln        3475 3480 3485 Lys Pro Val Val Ile Pro Gly Tyr Glu Gly Lys Thr Pro Leu Phe Gln    3490 3495 3500 Ile Phe Asp Lys Val Lys Lys Glu Trp Asp Gln Phe Gln Asn Pro Val 3505 3510 3515 3520 Ala Val Ser Phe Asp Thr Lys Ala Trp Asp Thr Gln Val Thr Thr Lys                3525 3530 3535 Asp Leu Glu Leu Ile Lys Asp Ile Gln Lys Tyr Tyr Phe Lys Lys Lys            3540 3545 3550 Trp His Lys Phe Ile Asp Thr Leu Thr Met His Met Ser Glu Val Pro        3555 3560 3565 Val Ile Ser Ala Asp Gly Glu Val Tyr Ile Arg Lys Gly Gln Arg Gly    3570 3575 3580 Ser Gly Gln Pro Asp Thr Ser Ala Gly Asn Ser Met Leu Asn Val Leu 3585 3590 3595 3600 Thr Met Val Tyr Ala Phe Cys Glu Ala Thr Gly Val Pro Tyr Lys Ser                3605 3610 3615 Phe Asp Arg Val Ala Lys Ile His Val Cys Gly Asp Asp Gly Phe Leu            3620 3625 3630 Ile Thr Glu Arg Ala Leu Gly Glu Lys Phe Ala Ser Lys Gly Val Gln        3635 3640 3645 Ile Leu Tyr Glu Ala Gly Lys Pro Gln Lys Ile Thr Glu Gly Asp Lys    3650 3655 3660 Met Lys Val Ala Tyr Gln Phe Asp Asp Ile Glu Phe Cys Ser His Thr 3665 3670 3675 3680 Pro Ile Gln Val Arg Trp Ser Asp Asn Thr Ser Ser Tyr Met Pro Gly                3685 3690 3695 Arg Asn Thr Thr Thr Ile Leu Ala Lys Met Ala Thr Arg Leu Asp Ser            3700 3705 3710 Ser Gly Glu Arg Gly Thr Ile Ala Tyr Glu Lys Ala Val Ala Phe Ser        3715 3720 3725 Phe Leu Leu Met Tyr Ser Trp Asn Pro Leu Ile Arg Arg Ile Cys Leu    3730 3735 3740 Leu Val Leu Ser Thr Glu Leu Gln Val Lys Pro Gly Lys Ser Thr Thr 3745 3750 3755 3760 Tyr Tyr Tyr Glu Gly Asp Pro Thr Ser Ala Tyr Lys Glu Val Ile Gly                3765 3770 3775 His Asn Leu Phe Asp Leu Lys Arg Thr Ser Phe Glu Lys Leu Ala Lys            3780 3785 3790 Leu Asn Leu Ser Met Ser Val Leu Gly Ala Trp Thr Arg His Thr Ser        3795 3800 3805 Lys Arg Leu Leu Gln Asp Cys Val Asn Met Gly Val Lys Glu Gly Asn    3810 3815 3820 Trp Leu Val Asn Ala Asp Arg Leu Val Ser Ser Lys Thr Gly Asn Arg 3825 3830 3835 3840 Tyr Ile Pro Gly Glu Gly His Thr Leu Gln Gly Arg His Tyr Glu Glu                3845 3850 3855 Leu Ala Leu Ala Arg Lys Gln Ile Asn Asn Phe Gln Gly Thr Asp Arg            3860 3865 3870 Tyr Asn Leu Gly Pro Ile Val Asn Met Val Leu Arg Arg Leu Arg Val        3875 3880 3885 Met Met Met Thr Leu Ile Gly Arg Gly Val    3890 3895 <210> 2 <211> 58 <212> PRT <213> Artificial Sequence <220> <223> ERNS-F1 <400> 2 Ala Gly Thr Val Ile Glu Gly Pro Cys Asn Phe Asn Val Ser Val Glu   1 5 10 15 Asp Ile Leu Tyr Gly Asp His Glu Cys Gly Ser Leu Leu Gln Asp Thr              20 25 30 Ala Leu Tyr Leu Val Asp Gly Met Thr Asn Thr Ile Glu Asn Ala Arg          35 40 45 Gln Gly Ala Ala Arg Val Thr Ser Trp Leu      50 55 <210> 3 <211> 174 <212> DNA <213> Artificial Sequence <220> <223> ERNS-F1 <400> 3 gcaggtacag ttatagaggg cccatgtaat ttcaatgttt ccgtggagga tatcttgtat 60 ggggatcatg agtgcggcag tttgctccag gacacggctc tgtacctagt agatggaatg 120 accaacacta tagagaatgc cagacaggga gcagcgaggg taacatcttg gctc 174 <210> 4 <211> 118 <212> PRT <213> Artificial Sequence <220> <223> ERNS-F2 <400> 4 Ala Gly Thr Val Ile Glu Gly Pro Cys Asn Phe Asn Val Ser Val Glu   1 5 10 15 Asp Ile Leu Tyr Gly Asp His Glu Cys Gly Ser Leu Leu Gln Asp Thr              20 25 30 Ala Leu Tyr Leu Val Asp Gly Met Thr Asn Thr Ile Glu Asn Ala Arg          35 40 45 Gln Gly Ala Ala Arg Val Thr Ser Trp Leu Glu Phe Ala Gly Thr Val      50 55 60 Ile Glu Gly Pro Cys Asn Phe Asn Val Ser Val Glu Asp Ile Leu Tyr  65 70 75 80 Gly Asp His Glu Cys Gly Ser Leu Leu Gln Asp Thr Ala Leu Tyr Leu                  85 90 95 Val Asp Gly Met Thr Asn Thr Ile Glu Asn Ala Arg Gln Gly Ala Ala             100 105 110 Arg Val Thr Ser Trp Leu         115 <210> 5 <211> 354 <212> DNA <213> Artificial Sequence <220> <223> ERNS-F2 <400> 5 gcaggtacag ttatagaggg cccatgtaat ttcaatgttt ccgtggagga tatcttgtat 60 ggggatcatg agtgcggcag tttgctccag gacacggctc tgtacctagt agatggaatg 120 accaacacta tagagaatgc cagacaggga gcagcgaggg taacatcttg gctcgaattc 180 gcaggtacag ttatagaggg cccatgtaat ttcaatgttt ccgtggagga tatcttgtat 240 ggggatcatg agtgcggcag tttgctccag gacacggctc tgtacctagt agatggaatg 300 accaacacta tagagaatgc cagacaggga gcagcgaggg taacatcttg gctc 354 <210> 6 <211> 178 <212> PRT <213> Artificial Sequence <220> <223> ERNS-F3 <400> 6 Ala Gly Thr Val Ile Glu Gly Pro Cys Asn Phe Asn Val Ser Val Glu   1 5 10 15 Asp Ile Leu Tyr Gly Asp His Glu Cys Gly Ser Leu Leu Gln Asp Thr              20 25 30 Ala Leu Tyr Leu Val Asp Gly Met Thr Asn Thr Ile Glu Asn Ala Arg          35 40 45 Gln Gly Ala Ala Arg Val Thr Ser Trp Leu Glu Phe Ala Gly Thr Val      50 55 60 Ile Glu Gly Pro Cys Asn Phe Asn Val Ser Val Glu Asp Ile Leu Tyr  65 70 75 80 Gly Asp His Glu Cys Gly Ser Leu Leu Gln Asp Thr Ala Leu Tyr Leu                  85 90 95 Val Asp Gly Met Thr Asn Thr Ile Glu Asn Ala Arg Gln Gly Ala Ala             100 105 110 Arg Val Thr Ser Trp Leu Lys Leu Ala Gly Thr Val Ile Glu Gly Pro         115 120 125 Cys Asn Phe Asn Val Ser Val Glu Asp Ile Leu Tyr Gly Asp His Glu     130 135 140 Cys Gly Ser Leu Leu Gln Asp Thr Ala Leu Tyr Leu Val Asp Gly Met 145 150 155 160 Thr Asn Thr Ile Glu Asn Ala Arg Gln Gly Ala Ala Arg Val Thr Ser                 165 170 175 Trp Leu         <210> 7 <211> 534 <212> DNA <213> Artificial Sequence <220> <223> ERNS-F3 <400> 7 gcaggtacag ttatagaggg cccatgtaat ttcaatgttt ccgtggagga tatcttgtat 60 ggggatcatg agtgcggcag tttgctccag gacacggctc tgtacctagt agatggaatg 120 accaacacta tagagaatgc cagacaggga gcagcgaggg taacatcttg gctcgaattc 180 gcaggtacag ttatagaggg cccatgtaat ttcaatgttt ccgtggagga tatcttgtat 240 ggggatcatg agtgcggcag tttgctccag gacacggctc tgtacctagt agatggaatg 300 accaacacta tagagaatgc cagacaggga gcagcgaggg taacatcttg gctcaagctt 360 gcaggtacag ttatagaggg cccatgtaat ttcaatgttt ccgtggagga tatcttgtat 420 ggggatcatg agtgcggcag tttgctccag gacacggctc tgtacctagt agatggaatg 480 accaacacta tagagaatgc cagacaggga gcagcgaggg taacatcttg gctc 534 <210> 8 <211> 186 <212> DNA <213> Artificial Sequence <220> <223> pET23a (+) ERNS-F1 <400> 8 ggatccgcag gtacagttat agagggccca tgtaatttca atgtttccgt ggaggatatc 60 ttgtatgggg atcatgagtg cggcagtttg ctccaggaca cggctctgta cctagtagat 120 ggaatgacca acactataga gaatgccaga cagggagcag cgagggtaac atcttggctc 180 gaattc 186 <210> 9 <211> 62 <212> PRT <213> Artificial Sequence <220> <223> pET23a (+) ERNS-F1 <400> 9 Gly Ser Ala Gly Thr Val Ile Glu Gly Pro Cys Asn Phe Asn Val Ser   1 5 10 15 Val Glu Asp Ile Leu Tyr Gly Asp His Glu Cys Gly Ser Leu Leu Gln              20 25 30 Asp Thr Ala Leu Tyr Leu Val Asp Gly Met Thr Asn Thr Ile Glu Asn          35 40 45 Ala Arg Gln Gly Ala Ala Arg Val Thr Ser Trp Leu Glu Phe      50 55 60 <210> 10 <211> 26 <212> DNA <213> Artificial Sequence <220> <223> ERNS-F2 sense <400> 10 cggaattcgc aggtacagtt atagag 26 <210> 11 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> ERNS-F2 anti-sense <400> 11 caagcttgag ccaagatgtt accct 25 <210> 12 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> ERNS-F3 sense <400> 12 cccaagcttg caggtacagt tatagag 27 <210> 13 <211> 26 <212> DNA <213> Artificial Sequence <220> <223> ERNS-F3 anti-sense <400> 13 ccctcgagga gccaagatgt taccct 26 <210> 14 <211> 189 <212> DNA <213> Artificial Sequence <220> <223> PCR-ERNS-F2 <400> 14 cggaattcgc aggtacagtt atagagggcc catgtaattt caatgtttcc gtggaggata 60 tcttgtatgg ggatcatgag tgcggcagtt tgctccagga cacggctctg tacctagtag 120 atggaatgac caacactata gagaatgcca gacagggagc agcgagggta acatcttggc 180 tcaagcttg 189 <210> 15 <211> 366 <212> DNA <213> Artificial Sequence <220> <223> pET23a (+) ERNS-F2 <400> 15 ggatccgcag gtacagttat agagggccca tgtaatttca atgtttccgt ggaggatatc 60 ttgtatgggg atcatgagtg cggcagtttg ctccaggaca cggctctgta cctagtagat 120 ggaatgacca acactataga gaatgccaga cagggagcag cgagggtaac atcttggctc 180 gaattcgcag gtacagttat agagggccca tgtaatttca atgtttccgt ggaggatatc 240 ttgtatgggg atcatgagtg cggcagtttg ctccaggaca cggctctgta cctagtagat 300 ggaatgacca acactataga gaatgccaga cagggagcag cgagggtaac atcttggctc 360 aagctt 366 <210> 16 <211> 122 <212> PRT <213> Artificial Sequence <220> <223> pET23a (+) ERNS-F2 <400> 16 Gly Ser Ala Gly Thr Val Ile Glu Gly Pro Cys Asn Phe Asn Val Ser   1 5 10 15 Val Glu Asp Ile Leu Tyr Gly Asp His Glu Cys Gly Ser Leu Leu Gln              20 25 30 Asp Thr Ala Leu Tyr Leu Val Asp Gly Met Thr Asn Thr Ile Glu Asn          35 40 45 Ala Arg Gln Gly Ala Ala Arg Val Thr Ser Trp Leu Glu Phe Ala Gly      50 55 60 Thr Val Ile Glu Gly Pro Cys Asn Phe Asn Val Ser Val Glu Asp Ile  65 70 75 80 Leu Tyr Gly Asp His Glu Cys Gly Ser Leu Leu Gln Asp Thr Ala Leu                  85 90 95 Tyr Leu Val Asp Gly Met Thr Asn Thr Ile Glu Asn Ala Arg Gln Gly             100 105 110 Ala Ala Arg Val Thr Ser Trp Leu Lys Leu         115 120 <210> 17 <211> 191 <212> DNA <213> Artificial Sequence <220> PCR-ERNS-F3 <400> 17 cccaagcttg caggtacagt tatagagggc ccatgtaatt tcaatgtttc cgtggaggat 60 atcttgtatg gggatcatga gtgcggcagt ttgctccagg acacggctct gtacctagta 120 gatggaatga ccaacactat agagaatgcc agacagggag cagcgagggt aacatcttgg 180 ctcctcgagg g 191 <210> 18 <211> 546 <212> DNA <213> Artificial Sequence <220> <223> pET23a (+) ERNS-F3 <400> 18 ggatccgcag gtacagttat agagggccca tgtaatttca atgtttccgt ggaggatatc 60 ttgtatgggg atcatgagtg cggcagtttg ctccaggaca cggctctgta cctagtagat 120 ggaatgacca acactataga gaatgccaga cagggagcag cgagggtaac atcttggctc 180 gaattcgcag gtacagttat agagggccca tgtaatttca atgtttccgt ggaggatatc 240 ttgtatgggg atcatgagtg cggcagtttg ctccaggaca cggctctgta cctagtagat 300 ggaatgacca acactataga gaatgccaga cagggagcag cgagggtaac atcttggctc 360 aagcttgcag gtacagttat agagggccca tgtaatttca atgtttccgt ggaggatatc 420 ttgtatgggg atcatgagtg cggcagtttg ctccaggaca cggctctgta cctagtagat 480 ggaatgacca acactataga gaatgccaga cagggagcag cgagggtaac atcttggctc 540 ctcgag 546 <210> 19 <211> 182 <212> PRT <213> Artificial Sequence <220> <223> pET23a (+) ERNS-F3 <400> 19 Gly Ser Ala Gly Thr Val Ile Glu Gly Pro Cys Asn Phe Asn Val Ser   1 5 10 15 Val Glu Asp Ile Leu Tyr Gly Asp His Glu Cys Gly Ser Leu Leu Gln              20 25 30 Asp Thr Ala Leu Tyr Leu Val Asp Gly Met Thr Asn Thr Ile Glu Asn          35 40 45 Ala Arg Gln Gly Ala Ala Arg Val Thr Ser Trp Leu Glu Phe Ala Gly      50 55 60 Thr Val Ile Glu Gly Pro Cys Asn Phe Asn Val Ser Val Glu Asp Ile  65 70 75 80 Leu Tyr Gly Asp His Glu Cys Gly Ser Leu Leu Gln Asp Thr Ala Leu                  85 90 95 Tyr Leu Val Asp Gly Met Thr Asn Thr Ile Glu Asn Ala Arg Gln Gly             100 105 110 Ala Ala Arg Val Thr Ser Trp Leu Lys Leu Ala Gly Thr Val Ile Glu         115 120 125 Gly Pro Cys Asn Phe Asn Val Ser Val Glu Asp Ile Leu Tyr Gly Asp     130 135 140 His Glu Cys Gly Ser Leu Leu Gln Asp Thr Ala Leu Tyr Leu Val Asp 145 150 155 160 Gly Met Thr Asn Thr Ile Glu Asn Ala Arg Gln Gly Ala Ala Arg Val                 165 170 175 Thr Ser Trp Leu Leu Glu             180 <210> 20 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> candidate 1 <400> 20 Ala Gly Thr Val Ile Glu Gly Pro   1 5 <210> 21 <211> 15 <212> PRT <213> Artificial Sequence <220> <223> candidate 2 <400> 21 Asn Phe Asn Val Ser Val Glu Asp Ile Leu Tyr Gly Asp His Glu   1 5 10 15 <210> 22 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> candidate 3 <400> 22 Gly Ala Ala Arg Val Thr Ser Trp Leu   1 5

Claims (18)

An amino acid sequence of SEQ ID NO: 20 located at 414 to 421th from SEQ ID NO: 1, SEQ ID NO: 21 located at 423 to 437, and amino acid sequence of SEQ ID NO: 22 located at 463 to 471th of the amino acid sequence of SEQ ID NO: 1 Repeating two to five times in series with an antigen-derived peptide consisting of 58 to 68 amino acids,
Fusion peptides specific for antibodies to swine fever virus. The method of claim 1,
The antigen-derived peptide is a fusion peptide consisting of the amino acid sequence of SEQ ID NO: 2. The fusion peptide according to claim 2, wherein the amino acid sequence of SEQ ID NO: 2 is encoded by the nucleotide sequence of SEQ ID NO: 3. The method of claim 1,
The fusion peptide is a fusion peptide consisting of the amino acid sequence of SEQ ID NO: 4 or SEQ ID NO: 6. The fusion peptide of claim 4, wherein the amino acid sequence of SEQ ID NO: 4 is encoded by the nucleotide sequence of SEQ ID NO: 5. The fusion peptide of claim 4, wherein the amino acid sequence of SEQ ID NO: 6 is encoded by the nucleotide sequence of SEQ ID NO: 7. 6. An amino acid sequence of SEQ ID NO: 20 located at 414 to 421th from SEQ ID NO: 1, SEQ ID NO: 21 located at 423 to 437, and amino acid sequence of SEQ ID NO: 22 located at 463 to 471th of the amino acid sequence of SEQ ID NO: 1 Antigen-derived peptide consisting of 58 to 68 amino acids or repeating the peptide two to five times in series,
Composition for detecting swine fever virus infection. Claim 7 swine fever virus infection detection kit comprising a composition for detecting swine fever virus infection. The kit of claim 8, wherein the kit comprises a catalyst, an enzyme, an enzyme substrate, a fluorescent compound, a chemiluminescent compound, a metal sol, a dye sol, a particulate latex, and a color indicator. , Detection kit further comprising one or more labeling reagents selected from the group consisting of color matter, carbon sol and selenium sol contained in liposomes. The detection kit of claim 8, wherein the kit detects an ERNS antibody against infection of porcine fever virus using an antigen-antibody reaction between a specimen and the infection detection composition. The detection kit of claim 10, wherein the sample is blood, serum, plasma, lymph, tissue, urine, tear, saliva, milk, vomiting, or feces. The detection kit of claim 8, wherein the kit is in the form of an enzyme-linked immunosorbent assay (ELISA) kit, a blotting kit, an immunoprecipitation kit, an immunofluorescence kit or an immune strip. The method of claim 12, wherein the detection kit in the form of an immune strip
A sample pad absorbing a sample;
A membrane showing a detection result; And
A detection kit comprising one or more assay strips comprising an absorbent pad that absorbs sample development. The method of claim 13,
The detection kit in the form of an immunostrip has two or more assay strips,
Any one of the above analysis strips is SEQ ID NO: 20 located at 414 to 421th from SEQ ID NO: 1, SEQ ID NO: 21 located at 423 to 437, and amino acid of SEQ ID NO: 22 located at 463 to 471 of the amino acid sequence of SEQ ID NO: 1 An antigen-derived peptide consisting essentially of 58 to 68 consecutive amino acids, including a sequence, or a membrane to which a fusion peptide comprising the peptide repeats two to five times in series is immobilized,
Another assay strip comprises a membrane to which additional antigens or antibodies are immobilized. The method of claim 13, wherein the detection kit in the form of an immune strip
A lower case supporting one side of the analysis strip; And
An upper case coupled to the lower case and covering the other surface of the assay strip, the upper case including a sample inlet corresponding to the sample pad, a result display window corresponding to the membrane, and a character display corresponding to the absorption pad;
It further comprises a detection kit. A method of discriminating a vaccine strain or an outdoor strain of swine fever virus using the composition for detecting swine fever virus infection of claim 7 or the detection kit of claim 8. The method of claim 16,
The vaccine strain is a purified lively toxic vaccine strain or a recombinant marker vaccine strain. Preparing a sample;
The amino acid sequence of SEQ ID NO: 20 located at 414 to 421th from SEQ ID NO: 1, SEQ ID NO: 21 located at 423 to 437th, and SEQ ID NO: 22 located at 463 to 471th of the amino acid sequence of SEQ ID NO: 1 are essential to the specimen. Applying an antigen-derived peptide consisting of 58 to 68 consecutive amino acids, or a fusion peptide comprising two to five repetitions of the peptide in series; And
Examining an antigen-antibody reaction between the sample and the antigen-derived peptide or the fusion peptide
Including, the antibody detection method for swine fever virus.

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