The Expression And Application Of The Main Antigenic Region Of VP2 Protein Of PPV

Porcine parvovirus(PPV) is one of the major fectors which causes mummification, still births and other reproductive failures in pregnant sows. It causes lots of economical losses in swine production. At present, there is no efficient diagnostic kit in the domestic market. The rapid and specific diagnostic method is much important to control these diseases. The VP2 protein of PPV is a major structural protein of virus and has specific immunogenicity.The VP2 gene is a conserved sequence in the viral genome. The expressed product would be used in diagnosis. So as follows in this research are to set up the diagnostic method on the basis of VP2 protein and protect against the diseases associated with PPV.1. We designed one pairs of primers and amplified the VP2 genes from the vector PET-VP2 I which was constructed by LiTing, and the PCR products were inserted into secretory pPICZα-A. The recombinant plasmid pPICZα-A-VP2 I with VP2 gene were acquired and induced with methanol, SDS-PAGE and Western blot analysis showed that the gene could be expressed in P. pastoris and the products had good reacting ability.2. We studied the relations between expression yield and growth conditions and found the optimal expression conditions of the recombinant VP2 protein were: 28-30℃, 225rpm, when BMGY with OD₆₀₀ being 3, the recombinant Picha Pastoris was add to the BMMY medium of pH7.0 which with OD₆₀₀ being 1.5. Then the solution was incubated 72h and methanol was added to keep its concentration up to 1.0% between 24h. After induction under optimal conditions, the yield of VP2 protein could be added up to 227.6ug/mL. It was found that the supernatant should be conserved in -20℃ after comparing effects of different conservation temperature on lysisof protein VP2.3. An indirect ELISA using protein VP2 which was expressed by Picha Pastoris as coating antigen was established to monitor antibodies level of pigs against PPV and its operation methods were optimized. 200 pieces of sera from pigs infected by PPV were examined by this method and compated with the method of latex aggllutination diagnostic kit which constructed by HuaZhong Agricultural university. The agreement ratio between the two methods was 92.6%.4. The recombinant plasmid PET-VP2 I was transformed to BL21 competent cell and expessed in very high level after induced with 1.0mmol/l IPTG.The expressed product was...