Expression Of Classical Swine Fever Virus E2 Protein And The Preparation Of Monoclone Antibody

Classical swine fever (CSF) is a acute infectious disease caused by the Classical swine fever virus (CSFV), and its main symptoms is fever and bleeding. Classical swine fever is distributed almost world-wide, spread rapidly, clinical symptoms multiplicity, and is considered the most economically significant disease of swine. Classical swine fever was classified as a class infectious disease by the world organization for animal health (OIE), one class infectious diseases by China. CSFV genome encoding four structural protein, which the E2 protein can induce the body to produce protective antibody during virus infection, resistance virus invasion, so E2 protein is the target protein to establish the diagnosis method of CSF, and vaccine preparating.Through the analysis of the nucleic sequence of E2 gene, design six pairs of primers. We get E2 gene which include CSFV itself signal peptide in N-terminal, and six his amino acids in C-terminal, get HE2 gene at the same time which include honeybee melittin signal peptide in N-terminal, and six his amino acids in C-terminal. General connecting clone genes to the pMD-18T vector, get pMD18-HE2 and pMD18-E2 plasmid, through the sequencing of nucleotide sequences both are right. Insert genes fragment to transfer vector pFastBac-eGFP-HA-NA-M1, obtain restructuring transfer plasmid of pFBD-3HE2 and pFBD-3E2. Conversion the recombinant plasmid into DH10Bac competent cells, extract rod granule through the blue-white selection, transfection sf9 cells, to obtain recombinant baculovirus vBac-3HE2 and vBac-3E2. Through Western blot test showed that the E2 protein successfully expressed in insect cells.Designed and synthesized two antigen peptides to aim directly at different antigen site by analyzing the antigen of E2 gene antigen site, immunization 4-5 weeks of BALB/C mice, collection blood at 10 days after third immunization, capture serum, detection antibody titer in the serum, take mice spleen cells which the antibody titer achieve the requirement, then carry out cell fusion, screening the cells of clone antibody secretion positive rate as 100% through the ELISA method, establish positive cell lines. Using CSF E2 protein to detect by Western blot and indirect immunofluorescence (IFA), finally obtain the six strains mAbs that can react with specificity of E2 proteins, named 1F7,1F5,2F11,2E6,2F5,2H2 respectively. Our study laid a foundation for the establishment of the CSF antibody detection method.