KR20070116382A - Swine fever virus erns protein and method for screening and detecting a swine fever virus antibody that spcifically reacts with the same - Google Patents

Abstract

A method for screening and detecting a swine fever virus antibody is provided to easily screen and diagnose infected swine fever virus antibody by using a monoclonal antibody specifically reacting with a recombinant swine fever virus Erns protein and secure constant sensitivity regardless of all genotypes of the swine fever virus when using all three kinds of recombinant protein. A recombinant swine fever virus Erns is prepared by expressing a gene encoding three Erns proteins among swine fever viruses having various genotypes using baculovirus. A method for preparing a monoclonal antibody specifically reacting to the Erns comprises the steps of: (a) constructing a swine fever virus LOM strain-derived Erns recombinant gene using a baculovirus expression system; and (b) producing the monoclonal antibody using the recombinant Erns protein. A method for screening and detecting a swine fever virus antibody comprises the steps of: (a) mixing three kinds of Erns recombinant proteins at proper concentration to prepare an antigen cocktail; (b) after diluting the antigen cocktail into a coating buffer solution, pouring it in a plate to be absorbed into the plate; (c) washing the recombinant antigen not-absorbed into the plate to remove it; (d) adding a sample to be tested to the plat to allow the reaction; (e) washing the sample to be tested not specifically bound to the recombinant Erns antigen protein absorbed into the plate to remove it; (f) adding a monoclonal antibody not being bound to the enzyme but being specifically reacted to the Erns protein to allow the reaction; (g) washing the monoclonal antibody not being bound to the recombinant Erns antigen protein absorbed into the plate to remove it; and (h) adding a substrate reacting with the enzyme to determine the swine fever virus infection.

Description

SWORE FEVER VIRUS ERNS PROTEIN AND METHOD FOR SCREENING AND DETECTING A SWINE FEVER VIRUS ANTIBODY THAT SPCIFICALLY REACTS WITH THE SAME}

1 is a flow chart schematically showing a method for diagnosing porcine cholera virus antibody using a recombinant protein and a monoclonal antibody of the present invention.

2 is a photograph showing the result of protein fractionation according to the present invention.

In the present invention, three types of E ^rns extracted from hog cholera virus of various genotypes (CSFV type 1, type 2 and type 3) isolated from Korea (Hog cholera virus, HCV. Classical swine fever virus, CSFV) The present invention relates to a method for diagnosing porcine cholera using a recombinant protein expressed in a baculovirus and a monoclonal antibody specific for the protein, and genotyping using a blocking enzyme linked immunosorbent assay (Blocking Enzyme Linked Immunosorbent Assay; Blocking-ELISA). The present invention relates to a diagnostic method capable of detecting swine cholera antibody irrespective of the condition and rapidly discriminating and testing the antibody by field infection when the gene-engineered marker vaccine of swine cholera virus is used.

Swine cholera is a contagious disease that is infected by the swine cholera virus. Pigs are the only natural host, and in the case of acute swine cholera, high fever, skin flare, loss of appetite, constipation, diarrhea, white blood cell reduction, posterior palsy, and similar acids It is a disease with high mortality and morbidity accompanied with such symptoms. In Korea, it is classified as a type 1 statutory livestock epidemic, and the OIE classifies it as a list A disease.

Porcine cholera virus is an RNA virus belonging to Flaviviridae (Pestivirus), the size of the virus is known to be 12.3 to 12.5Kb. Genomic RNA consists of 5'-enoncoding reaction (NCR), single open reading frame (ORF) and 3'-NCR. A single ORF is cleaved into the virus's structural proteins capsid C, E ^rns , E1 and E2, respectively. In addition, it is cleaved into N ^pro , p7, NS2-3, NS4A, NS4B, NS5A, NS5B as nonstructural proteins, respectively. E2 protein is a major structural protein of porcine cholera virus and a protein that induces a virus neutralizing antibody reaction, and is known immunologically to play an important role in the defense mechanism of porcine cholera. In Europe, marker vaccines using E2 are being studied and their use is recommended under certain conditions.

In Korea, LOM strain, a pure cholera purified live venom virus, has been produced and vaccinated as a live venom vaccine. However, the policy for eradicating swine cholera is focused on the industrialization of recombinant marker vaccines using E2 protein. Establishing. However, when a recombinant marker vaccine is used, a test method for distinguishing a vaccine antibody from an outdoor infection antibody has not been established. Therefore, it is expected to be difficult for the screening test after using the vaccine. Therefore, there is a need for the development of a differentiation kit that can distinguish between antibodies caused by outdoor readout porcine cholera virus and marker vaccine inoculated antibodies.

E ^rns , a structural protein of porcine cholera virus, is known to be the second epitope that induces neutralizing antibody response along with E2 and induces an immune response when the virus is infected. Therefore, when the marker vaccine is inoculated, other antibodies (E ^rns ) of porcine cholera are formed in addition to the antibody to E2. Therefore, if an antibody other than the antibody to E2 can be detected, the infected animal can be quickly discriminated.

E ^rns of porcine cholera virus after marker vaccination already in foreign countries Differentiation methods using recombinant proteins have been evaluated and kits have been developed and commercialized by two companies (CEDI and CHECKIT), but these products have been reported to show inconsistent test results depending on the viral genotype. Therefore, there is an urgent need for the development of a differentiation kit that can show a certain test result for all genotypes of swine cholera virus.

Therefore, the present inventors conducted a study to develop a method capable of testing the E ^rns antibody regardless of all genotypes of porcine cholera virus, and as a result, 3 having various genotypes derived from three isolated swine cholera viruses in Korea E ^rns expresses a recombinant protein of the species and the E ^rns Monoclonal antibodies have been developed that exhibit specific reactivity to. The three recombination proteins were mixed to form antigens against them, and the specific monoclonal antibodies were used to overcome the differences in detection results according to genotypes through blocking ELISA.

An object of the present invention is to provide three types of genetic recombination proteins for diagnosing E ^rns antibodies of an animal infected with a swine cholera virus field strain, monoclonal antibodies thereof, a method for diagnosing swine cholera virus using the same, and an antibody against the recombinant protein. It is to provide a discrimination kit using.

In order to achieve the above object, the present invention provides three types of E ^rns of porcine cholera virus of various genotypes isolated in Korea. Recombinant porcine cholera virus E ^rns expresses the gene encoding the protein using baculovirus Provide protein.

In the present invention, the pig cholera virus may be selected from the group consisting of LOM strain, Cheolwon strain, Yongin strain.

The present invention also provides a method for producing a monoclonal antibody that specifically reacts with E ^rns of porcine cholera virus. Porcine cholera virus LOM strain-derived E ^rns using a baculovirus expression system to produce the monoclonal antibody Recombinant ^Enrns protein obtained by constructing a recombinant gene can be used.

In addition, the present invention uses porcine cholera using an enzyme immunoassay using a mixed antigen of ^Erns recombinant protein, and a monoclonal antibody that specifically recognizes the antigen to detect all antibodies against various genotypes of porcine cholera virus at the same time. As a method for differentially diagnosing an outdoor host virus antibody, a diagnostic method including the following steps is provided:

(a) E ^rns of porcine cholera virus Preparing an antigen cocktail by mixing three recombinant proteins at an appropriate concentration;

(b) diluting the prepared antigen cocktail in a coating buffer followed by aliquoting and adsorbing onto a plate;

(c) washing and removing the recombinant antigen not adsorbed on the plate;

(d) after step (c), adding a test sample to the plate and reacting it;

(e) after step (d), the recombinant ^Erns adsorbed on the plate Washing and removing the test sample that does not specifically bind the antigen protein;

(f) after step (e), adding a monoclonal antibody that does not bind to the enzyme but reacts specifically with the ^Erns protein;

(g) after step (f), the recombinant ^Erns adsorbed on the plate Washing and removing monoclonal antibodies that do not bind the antigenic protein;

(h) after step (g), determining whether the pig cholera virus is infected by adding a substrate that reacts with the enzyme.

In the present invention, it is preferable to use a competitive enzyme immunoassay as the enzyme immunoassay.

In addition, the present invention is the porcine cholera virus E ^rns described above Provided are porcine cholera differentiation diagnostic kits for testing antibodies to proteins.

Example

Hereinafter, the present invention will be described in detail by way of specific examples, but the following examples are only examples for demonstrating the constitution and effects of the present invention, and the present invention is not limited to the following examples.

Example 1: CSFV E ^rns protein manufactured by recombinant genotypes

(1) gene recombinant baculovirus production

Genes encoding E ^rns proteins from three genotypes of swine cholera viruses (types 1, 2 and 3) isolated from Korea were amplified by Reverse Transcription Polymerase Chain Reaction (RT-PCR). . The expression vector pAcgp67B-E ^rns was prepared by ligation of the amplified cDNA to pAcgp67B Baculovirus vector. Subsequently, the expression vector was transformed into E. coli DH5-α cells, and then cultured in agar palte to isolate the plasmid from the colonies formed. The plasmid was digested with restriction enzymes BamH1 and ECOR1, followed by the E ^rns gene. Was confirmed by electrophoresis. PAcgp67B-E ^rns After culturing a large amount of the introduced cells to secure a plasmid, sf 9 insect cells were subjected to Baculovirus linearized DNA and co-transfection to secure recombinant baculovirus.

(2) CSFV E ^rns Production of Recombinant Proteins

Infected cells were harvested after infecting the recombinant baculovirus with insect cells. Infected cells were lysed to elute the recombinant ^Erns protein. The recombinant protein was purified by combining the expressed E ^rns protein with Ni-NAT agarose (Qiagen, USA) and then adjusting the concentration of imidazole (Qiagen, USA). As a result of confirming the protein fraction using SDS-PAGE of the purified recombinant protein, E ^rns protein fraction (40-5-KDa) was identified as shown in FIG. 1. Three kinds of purified proteins were mixed at an appropriate concentration to prepare a mixed antigen for ELISA.

Example 2: Preparation of Hybridomas Producing Monoclonal Antibodies

The process of producing the hybridoma cell line producing monoclonal antibody is briefly as follows. DMEM (GibcoBRL, USA) medium supplemented with 10% fetal bovine serum after purchasing SP2 / O-Ag14, a myeloma cell line, from the Korea Collection for Type Culture (KCTC) Korea Biotechnology Center Inoculated at 5 ° C. and incubated at 37 ° C. in an incubator supplied with 5% carbon dioxide gas. Recombinant E ^{rns prepared} in Example 1 Protein antigens were homogenously mixed in the same amount with Incompleted Freund's Adjuvant, Sigma, USA to inoculate the muscles of 6-week-old BALB / C mice (Orient, Korea). Two and four weeks after immunization, the same amount of antigen was further immunized, and then spleens of the mice were aseptically collected and crushed, and only lymphocytes were recovered. Lymphocytes and tumor cells isolated were induced to fuse lymph cells with tumor cells using PEG 1500 (Sigma, USA). Then, the fused cells are appropriately diluted in HAT (Hypoxanthin Aminopterin Thymidine, GibcoBRL, USA) in DMEM selection medium added 10% to fetal bovine serum, and then the fused cells are dispensed in 96-well tissue culture plates. And incubated. After 2 weeks, the supernatant was examined, the cell line showing positive reaction was diluted, cloned in 96-well tissue culture plate, and the positive clone was selected to select a cell line specific for CSFV E ^rns , and the reactivity was confirmed by the following method. .

The purified three E ^rns recombinant proteins were diluted in carbonate bicarbonate buffer (Carbonate bicaonate buffer, Sigma USA), adsorbed onto ELISA plates, and reacted with positive control serum, open-air positive serum, and negative control serum. After the reaction it was reacted with monoclonal antibodies produced in selected cell lines and with anti mouse HRP conjugates. After the reaction, a color developing agent was added thereto to develop color and absorbance was measured after adding a color stop solution. In order to confirm the competition ability of the monoclonal antibody with the positive serum against the three antigens, the reaction rate was confirmed by calculating the competition reaction rate according to Equation 1 below.

Competition Reaction Rate (%) = 100-(absorbance of sample / absorbance of negative control × 100)

As a result, as shown in (Table 1), it was confirmed that the three E ^rns proteins showed a specific competitive reaction with the positive serum by more than 60%, and confirmed that they were competitively responding to the antigenic determination sites of the three antigens to which the positive serum reacted. It was.

Competition test result of monoclonal antibody ERNS-02 Manufacturing antigen sample Absorbance (OD) Competition reaction rate (%) E ^rns -LOM Positive control 0.969 72.6 Positive serum 0.828 76.5 Negative control 3.532 0.0 E ^rns -Yongin Positive control 0.685 62.5 Positive serum 0.742 59.4 Negative control 1.826 0.0 E ^rns -Cheorwon Positive control 0.529 62.3 Positive serum 0.537 61.7 Negative control 1.402 0.0

Example 3: Blocking ELISA using Recombinant ^Erns Protein and Specific Monoclonal Antibodies thereto

(1) Preparation of Enzyme (HRP) Conjugation with Monoclonal Antibody

Cell lines producing monoclonal antibodies were inoculated intraperitoneally of mice and ascites were harvested. The obtained ascites was purified using immunochromatography using protein A-coupled agarose gel (Affiprep Protein A agarose, BioRad, Germany), and then using the peroxidase conjugate kit (Peroxidase Conjugate Kit, Roche, Germany). Conjugation was completed by binding to monoclonal antibodies according to the manual.

(2) recombinant E ^rns Preparation of Mixed Antigens of Proteins

After expressing the three types of antigens expressed as described above to prepare each antigen, mixed antigens were prepared by mixing the three types of antigens at an appropriate concentration, which was used as an antigen for ELISA.

(3) Testing of Porcine Cholera Virus Antibodies Using Blocking ELISA

The three antigens prepared above were adsorbed onto the ELISA plate for each type, and the non-adsorbed antigens were removed by washing. Mixed antigens were also adsorbed onto the plate in the same manner. Phosphate buffer added with 3% bovine serum albumin was prepared, aliquoted into plates and allowed to react at room temperature. After the reaction, the serum of pigs infected with each genotype virus was diluted 1: 4 with dilution buffer, reacted on an antigen-adsorbed plate, and washed with a washing solution. The monoclonal antibody conjugated to the enzyme (HRP) was diluted to an appropriate dilution concentration, added to the plate, and then reacted again. After the washing reaction, the monoclonal antibody was reacted with color, and then absorbed by TMB coloring agent. The above results were determined by calculating the ratio of the absorbance of the test serum and the negative control absorbance (SN Ratio Value), and then positive when the SN value was 0.60 or less, and negative when the SN value was 0.60 or more. It was calculated by Equation 2.

SN ratio value (SN) = [average absorbance of test sample / average absorbance of negative control]

As a result of confirming the antigen reactivity, when the type 1 antigen was used as shown in Table 2, the virus was not detected with respect to the sera infected with the type 2 and type 3 viruses, and even when the type 2 and type 3 antigens were used. In addition, the virus was not detected in sera infected with type 2 and type 3 viruses. However, the detection sensitivity of the serum infected with type 1 virus was inferior, whereas when the mixed antigen was used, it was possible to detect the antibody of swine cholera virus whose sensitivity to the antibody was improved in all individuals.

Table 2 below shows the results of the evaluation of the effectiveness of each E ^rns recombinant antigen in the virus infection serum group.

As can be seen in Table 2, when E ^rns LOM is used as an antigen, in the test group inoculated with the LOM vaccine strain, antibodies were detected in all individuals 14 days after the inoculation, but in the test group inoculated with Cheorwon or Nonsan strains. After 21 days, no individuals were detected. In addition, when ^Erns Yong- ^Jin and ^Cheol-Won- Ju were used, no individuals were detected in the group inoculated with type 1 virus.

Table 3 below shows the results of evaluating the effectiveness of the ^Erns recombinant mixed antigen in the virus infected serogroup.

 As can be seen in Table 3, in the mixed antigen of the present invention, even after 28 days of inoculation, antibodies were detected in all subjects except one, indicating that the sensitivity was improved.

E ^rns when using the monoclonal antibody of the present invention Antibodies can be detected to easily discriminate between porcine cholera virus antibodies infected in the open air, and the diagnostic method of the present invention can be used for all genotypes of porcine cholera virus when three recombinant proteins are used simultaneously. Regardless of genotype, a certain sensitivity is shown, so that antibodies to porcine cholera virus can be easily detected.

Claims (6)

E ^rns of three genotypes of swine fever virus Recombinant porcine cholera virus E ^rns expresses the gene encoding the protein using baculovirus protein. According to claim 1, wherein the porcine cholera virus recombinant pig cholera virus E ^rns, characterized in that derived from the group selected from the group consisting of LOM strain, Cheolwon strain, Yongin strain protein. E ^rns of porcine cholera virus A method for producing monoclonal antibodies that specifically react to a method, wherein the method uses a baculovirus expression system to produce porcine cholera virus LOM strain-derived E ^rns. After producing a recombinant gene, a method of producing a monoclonal antibody using the obtained recombinant E ^rns protein. Porcine cholera out-of-pocket virus virus was synthesized by using an enzyme immunoassay using a mixed antigen of ^Erns recombinant protein and a monoclonal antibody that specifically recognizes the antigen to simultaneously detect all antibodies against various genotypes of porcine cholera virus. As a differential diagnosis method, the differential diagnosis method of porcine cholera outdoor strain virus antibody comprising the following steps: (a) E ^rns of porcine cholera virus Preparing an antigen cocktail by mixing three recombinant proteins at an appropriate concentration; (b) diluting the prepared antigen cocktail in a coating buffer followed by aliquoting and adsorbing onto a plate; (c) washing and removing the recombinant antigen not adsorbed on the plate; (d) after step (c), adding a test sample to the plate and reacting it; (e) after step (d), the recombinant ^Erns adsorbed on the plate Washing and removing the test sample that does not specifically bind the antigen protein; (f) after step (e), adding a monoclonal antibody that does not bind to the enzyme but reacts specifically with the ^Erns protein; (g) after step (f), the recombinant ^Erns adsorbed on the plate Washing and removing monoclonal antibodies that do not bind the antigenic protein; (h) after step (g), determining whether the pig cholera virus is infected by adding a substrate that reacts with the enzyme. 5. The method of claim 4, wherein the enzyme immunoassay is a competitive enzyme immunoassay. Porcine cholera virus E ^rns according to claim 1 Porcine cholera differential diagnostic kit for testing antibodies to proteins.

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