Expression Or ORF2Truncated Gene Of PCV2in Prokaryotic Cells And Preparation And Identification Of Monoclonal Antibodies Against Cap Protein Of PCV2

Based on the published capsid protein gene (ORF2) of PCV2strain，a pairof primers were designed and synthesized. The capsid(cap) protein truncated thenuclear localization signal was amplified by polymerase chain reaction (PCR), insertin expression plasmid carrier pET-32a (+) in accordance with predetermined readingframe, building recombination expression vector pET-32a-ORF2, then the vectorwere restructured into host bacterium BL21for expression of induction, Afterwestern-blot identification, fusion protein has specific biological activities was42kda.The two forms of recombinant protein prokaryotic expression (soluble Cap proteinsand inclusion bodies of Cap protein) of prokaryotic expression were purified, PCV2cytotoxicity be concentrated and dialyzed by PEG20000and be differentialcentrifμged to purified PCV2virus, but also the final purified soluble Cap proteinsand purified PCV2cytotoxicity were used as coating antigen to established two kindsof indirect ELISA for detection of anti-PCV2antibodies. After specificity andsensitivity tested, the result showed that the two indirect ELISA methods have highspecificity and sensitivity.And immuned BALB/c mice with PCV2cytotoxicity which was purified, as well as,purified Cap protein and PCV2was used as coated antigen, and established the twoindirect ELISA methods to screen and obtain two monoclonal antibody hybridomacell lines specific for PCV, the McAbs were named:1-A1,4-H11. The two hybridomacells inducted ascites antibody titer were1:204800,1:102400in the strains of mice.Two monoclonal antibody subclasses were IgG2b, the stability experiments of twohybridoma secreting anti-PCV2monoclonal antibody showed that hybridom secretedantibodies remain basically unchanged after continuous passage of10,20,25generations and two times of cryopreserved. This showed that stability of thehybridoma cell lines secred antibodies. And apply the1-H11monoclonal antibodies to established double antibody sandwich ELISA. After specific detected, there are notcross-reactivity with PRRS virus (PRRSV), porcine epidemic diarrhea virus (PEDV),porcine parvovirus (PPV), porcine Japanese encephalitis virus (JEV), porcinecircovirus type1(PCV1)); After sensitivity tested, it showed that the minimumdetectable concentration of PCV2is234.4ng/mL. And apply the double antibodysandwich ELISA method to test positive diseased material that belongs to ownerlaboratory for in line with the PCR method was92.3%.This methods provides a basisfor setting up a PCV2–testing which has high specificity and sensibility.