| An indirect ELISA method based on PCV2-Rep protein was established to investigate the prevalence of PCV2 and the dynamics of PCV2 antibody production in the swinery. In addition, an indirect ELISA of PCV1-Cap protein was also established to detect the infection of PCV1 in pigs.Recombinant plasmid pET-Rep2 and pET-Cap1 were obtained by gene synthesis, digestion and ligation. Then the recombinants were transformed into E. coli strain BL21 (DE3) for expression. SDS-PAGE electrophoresis showed that Rep2 expressed in the form of inclusion body was 40kDa approximately. And Cap1 protein expressed mainly in the form of soluble protein was 32kDa. Western Blot analysis showed that the recombinant protein Rep2 and Capl both exhibited antigenicity. The target proteins were purified by nickel affinity chromatography and the purified proteins both showed a purity of above 95%.According to the "The ELISA Guidebook", the purified recombinant protein Rep2 and Cap1 were used as the coating antigen in this ELISA to detect specific antibodies against PCV Rep2 and Capl protein, and the optimal dilution of antigen and serum, and other conditions were determined by checkerboard titration. This Rep2-ELISA, which has a good repeatability, sensitivity and specificity, is a promising tool for large scale monitoring of PCV2 antibodies in swinery. The indirect ELISA method of PCVl-Cap protein has high specificity and repeatability.For comparison, PCV2-Rep, PCV1-Cap and PCV2-Cap indirect ELISA methods were used to detect serum from pigs in different stages at the same time. The results showed that positive rate detected by PCV2-Rep indirect ELISA was 71.9% (527/816), by PCV2-Cap indirect ELISA was 81.9%(668/816), by PCV1-Cap indirect ELISA was 47.1%(216/459). The dynamics of antibodies against Rep2 and Cap2 showed that the antibody against Rep2 appeared 2 or 3 weeks earlier than the antibody against Cap2. Therefore, the Rep2-ELISA method is appropriate in the early detection of infection of PCV2. |
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