Expression Of The NS1 And VP2 Gene Of Porcine Parvovirus And Development Of Elisa Based On Recombinant Protein

The NS1 gene and VP2 gene of porcine parvovirus (PPV) of HN1-strain were amplified by polymerase chain reaction (PCR). The amplified NS1 fragment of PPV was 624bp in length ; the amplified VP2 fragment of PPV was 882bp in length . The NS1 or VP2 fragments and expression vector pGEX-4T-l were digested by the same restriction endonucleases. These genes were ligated and transformed into Escherichia coli. The insert position, the size and the reading frame all were corrected by PCR, restriction digestion and the sequence analysis. The result showed that the prokaryotic expression vectors were constructed successfully. Then the recombinants were transformed into BL21 (DE3) for gene expression with IPTG inducing. The expressed proteins were measured by SDS-PAGE and western-blotting. The results showed that the NS1 gene and VP2 gene can express successfully in E.coli. The western-blotting results indicated that the expressed proteins could be recognized by the PPV positive serum. These results showed that expressed proteins can be used in genetic engineering diagnostic antigen .The inclusion bodies were obtained by sonication of the bacterial cells, from which the purified activative expressed recombinant NS1 proteins and VP2 proteins were got by urea and Triton X-100 washing and by renaturing, refolding and dialyzing. The results of SDS-PAGE show that washing by urea and Triton X-100 can decrease the amounts of bacteria proteins in the inclusion body. The reactivity of purified protein was increased 30 times in ELISA experiments comparing with the routine antigen. The diagnostic antigen that is substitution of the whole viral antigen may be produced in batches. It is hopeful to develop the monoclonal antibody against NS1,VP2 protein further. Offered advanced technology for diagnosis of the disease of porcine parvovirus.Based on the purified recombinant proteins, an indirect ELISA method for detection of PPV antibodies was developed.The concentration of coating antigen of NS1 is 2.69μg/ml and VP2 is 2.29μg/ml, HRP labeled anti-porcine IgG (1: 40000) being incubated at 37□ for 1h. The ELISA assay was confirmed to have a good reiterativity, specificity and sensitivity by repeated test, crossing test and blocking...