The Study On NS1 And VP2 Gene Of Porcine Parvovirus

The PPV 7 909 was grown on PK-15 cell lines. The primiparous rabbits were challenged with the cell virus and gave stillbirth. The livers of stillborn foetus were homogenated. The genomic DNA of PPV was extracted by employing both the phenol/chloroform isolating protocol and boiling extract from the homogenate.The NS1 protein is a main non-structural protein of PPV, encoded by NS1 gene. It is well documented that PPV-infected animals generate specific antibody in a lasting period of time. Given that inactivated virus does not have NS1 protein, it is potential to develop an NS1-based diagnostic kit for identifying the inactivated-PPV vaccinated pigs and wild-type PPV infected pigs in the clinical settings. According to the literature, a pair of primers was designed and synthesized. The NS1 gene of PPV was amplified by PCR, and the resulting products were identified by agar gel electrophoresis. A specific band was shown at the position of 2.2 kb. The PCR products were purified by agar gel DNA purification and cloned into PET32(a). Then the cloned plasmids were transformed into E.coli JM109. The specific recombinant plaslnid was identified with two restriction endonucleases. The recombinant plasmid could be filtered and transformed into E. coli BL21. It was found that NS1 protein was expressed in E. coli BL21 at 6h after induction with IPTG. The target protein was shown on the gel of SDS-PAGE, a specific band could be seen on 90.0KD. The western-blotting results showed that expressed protein could be specifically bound by the PPV positive serum.The main structural protein of PPV, VP2, encoded by VP2 gene, harbours many potential epitopes of virus. It could be anticipated that a new nucleic acid vaccine would be developed in way of well-designated procedure. According to the literature, a pair of primers was designed and synthesized, the VP2 gene of PPV was amplified by PCR. The PCR products were checked by agar gel electrophoresis, a specificband could be seen on 1.8 kb. Then the VP2 and NS1 gene were cloned into the eukaryotic vector of pcDNA3.1 (+), resulting recombinant expression plasmid pcDNA-VP2, pcDNA-NS1. The two plasmids were used as DNA vaccine to immunize mice. The result showed that the pcDNA-VP2 plaslnid could induce neutralized antibody and the antibody efficiency is 1∶11, but the pcDNA-NS1 plasmids couldn't induced counteract antibody. These results show that VP2 gene can be strategically employed to develop new DNA vaccine.