| Porcine circovirus type 2 (PCV-2) is the causative agent of postweaning multisystemic wasting syndrome (PMMS) that has become an emerging and important disease in swine. The ORF2 gene of PCV-2 is the main region encoding the structural protein that can be use to differentiate PCV-1 and PCV-2.Based on the published PCV-2 sequences, a pair of clone primers which were specific to the ORF2 genes of PCV-2 were designed. A 597-by fragment was amplified by polymerase chain reaction(PCR). The DNA fragment was then successfully cloned into pGEM-T-Easy vector, which was confirmed by sequence analysis,PCR and restrict enzyme identification.Based on the ORF2 genes of PCV-2.a pair of express primers which were specific to the ORF2 genes of PCV-2 were designed. partition of the ORF2 of PCV-2 were cloned and inserted into the bacterial plasmid PQE30. After identification by PCR and restricted enzyme, the constructed recombinant expressed plasmids was transformed into the receptive cells of E.coli JM109 and induced IPTG. then the recombinant protein was highly expressed in the form of inclusion bodies.After the inclusion bodies of recombinant proteins were purified with washing buffer for several times, the recombinant proteins were purified. Following the purification of the recombinant proteins,Using the purified recombinant protein as coating antigen, an indirect ELISA was established for detection of antibody in serum to PCV2. ELISA kit was primarily prepared and applied for detection of antibodies in serum samples from piglets infected experimentally and swine farms.The various factors and conditions for ELISA were explored, and the optimal reaction conditions were determined. The ELISA had good specificity when compared with shijiyuanheng kit and Agricultural Univercity of the Center of the China kit. |
