Prokaryotic Expression of Porcine Circovirus Type 2 Capsid Protein and Preliminary Establishment of Indirect ELISA Antibody Detection Method
Cap protein,as the main structural protein of Porcine circovirus type 2(PCV2),constitutes the nucleocapsid of the virus and is the main immune protective antigen of PCV2,which has important significance in the serological diagnosis of PCV2.In this study,specific primers were designed according to the ORF2 gene sequence of PCV2,and the ORF2 gene of nuclear de-localization signal peptide was amplified by PCR,and cloned into the prokaryotic expression vector pCold-Ⅰthrough homologous recombination.The recombinant plasmid pCold-Ⅰ-Cap was successfully obtained after sequencing identification.The recombinant plasmid was transformed into the competent cell BL21 for expression.The expression of Cap protein was detected by Coomassie bright blue staining and Western blot assay.At the same time,an indirect ELISA method was established to detect PCV2 serum antibodies by optimizing reaction conditions.The results showed that the recombinant de-localization signal peptide Cap protein was soluble and could specifically bind to PCV2 positive serum.An indirect ELISA method was preliminarily established by exploring different protein envelope concentrations and different primary antibody incubation concentrations,thus laying a foundation for effective monitoring of PCV2 antibodies.
Porcine circovirus type 2Cap proteinhomologous recombinationprotein expression and purificationELISA
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