| The disease of porcine circovirus, associated with postweaning multisystemic wasting syndrome(PMWS), porcine dermatitis and nephropathy syndrome(PDNS)and reproductive failures, etc., is aninfectious disease of porcine caused by porcine circovirus serotype 2(PCV2). Furthermore, the immunesystem of infectious pigs is inhibited by PCV2, easily secondary infecting other pathogens andexacerbating the disease. To provide easy and rapid test method and tool for the clinical diagnosis andepidemiological investigation of PCV2, the capsid protein (Cap protein) of PCV2 was expressed byprokaryotic expression system, purified by nickel affinity chromatography, and used as antigen ofindirect ELISA, the indirect ELISA method was researched and a detecting kit was developed by theoptimal reaction conditions of EL1SA.The recombinant expression and immunogenicity analysis of PCV2 Cap protein: Because the Capprotein encoded by ORF2 gene has the most antigenic epitopes of PCV2, the part of ORF2 gene,deleted the gene sequence of signal peptides in amino terminal of Cap protein and amplified by a pair ofspecific primers, was cloned into pET-32a(+) expression vector and inductive expressed by IPTG inE.coli BL21(DE3). The results of SDS-PAGE and Western-blotting using the virus-specific antibodiesshowed that, the recombinant protein was 40ku, and its good reactionogenicity could be used as coatingantigen for indirect ELISA method.The purification of PCV2 Cap recombinant protein and the development of indirect ELISA method:the yield of PCV2 Cap recombinant protein was about 20.71% of the total proteins after optimizing theexpression conditions(induction temperature, induction time and IPTG concentration were30℃,6h,1mmol/L,respectively), the density of PCV2 Cap recombinant protein purified by nickelaffinity chromatography reached 679.34μg/mL. Using the purified Cap recombinant protein as coatingantigen, the reaction conditions of ELISA were optimized. The coating concentration of protein was4μg/mL, incubated for 1 h at 37℃and then overnight at 4℃. Subsequently, the plates were blockedwith 5% calf serum overnight at 4℃, and then incubated for 1 h at 37℃with diluted serum sample(1:100) and diluted HRP-SPA (1:8000) respectively, and color developing time was incubated with 5min at 37℃. The cut-off value of ELISA was determined by detecting 30 portions PCV2 negative serum.The threshold for ELISA was OD450nm＞0.3280 for positives, OD450nm <0.2868 for negatives, and theother was suspicious. In comparative experiments with commercial ELISA Kit for detection of 55serum samples, the sensitivity, specificity and coincidence rate of the established ELISA method were95.12%,92.86% and 94.55% respectively. The established ELISA method had no cross-reactivity withthe reference sera of other swine viruses(PCV1, CSFV, PRRSV, PPV, PRV).The development and primary application of ELISA antibody detection kit for PCV2: the ELISAantibody detection kit was developed by the optimal reaction conditions of EL1SA. The intra-assayvariation coefficient was 2.3%-4.5%, interassay variation coefficient was 1.12%-2.63% respectively.The ELISA antibody detection kit could be stable at -20℃at least for one year. 66 serum samples ofthree pig farms from Anhui, Guangdong, Guangxi were detected by detection kit, and the positive rate was 100%, 48.39%, 100% respectively.The study have developed an inexpensive, rapid, specific diagnostic method,which provides aavailable technique for the serological detection and epidemiological survey of PCV2 and a technicalfundament for the further development and commercialization of the kit．...
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