This experiment was conducted to establish indirect ELISA using Cap protein of porcine arcovirus type2 (PCV2) as coating antigen The Rosetta E. coli with recombinant plasmid pET32a-ORF2 contain PCV2 ORF2 gene was induced by 1. 0 mmol · L-1 IPTG at 37 ℃. The indirect ELISA for detecting PCV2 Cap protein antibody was established to determine the immunogeniaty of the recombinant protein which was proved by Western blot. The optimum working circumstances for the ELISA are as follows : optimal concentration of Cap for coating was 2. 16 μg · mL-1 ; the dilution of serum sample was 1:40; the optimal coating condition of Cap for ELISA was incubated at 4 ℃ ovemight; the best blocking condition was in 1％ BSA at 37 ℃ for 1 h; the best incubation period and temperature for the serum sample and HRP-Iabeled rabbit anti-porcine IgG was 37 ℃ for 30 min and 37 ℃ for 45 min, respectively;the substrate for ELISA was incubated at 37 ℃ for 15 min before being terminated with the stopping solution 184 sera samples were examined with this ELISA in Sichuan province. The total seropositive is 80. 98％ (149/184) ,which is similar to commercial ELISA kits. The results indicated that the indirect ELISA is not only sensitive and specific, but also suitable for large-scale epidemiological investigation for PCV2 infection%本研究旨在建立以PCV2 Cap蛋白为包被抗原的间接ELISA检测方法,构建猪圆环病毒2型(PCV2)ORF2基因原核表达质粒pET32a-ORF2并转化至Rosetta菌,在1.0 mmol·L-1 IPTG和37℃条件下诱导下,Cap蛋白获得高效表达,Western blot检测证实其具有免疫反应活性,以纯化的蛋白为抗原建立了检测猪圆环病毒2型抗体的间接ELISA方法.结果表明抗原最适包被浓度为2.16μg·mL-1;血清最佳稀释度为1:40;抗原最佳包被条件为4℃过夜;最佳封闭条件为1%BSA 37℃1 h;血清反应时间为37℃30 min;酶标二抗最适作用时间为37℃45 min;底物最佳反应条件为37℃显色15 min.用该方法对四川省184份猪血清样品进行检测,总阳性率为80.98%(149/184),与商品化的PCV2 ELISA试剂盒得到的结果基本一致.本试验成功建立了PCv2血清抗体间接ELISA方法,具有较高的敏感性和特异性,可用于大规模的血清学检测.
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