Expression Of The ORF2 Gene Of PCV-2 In Pichia Pastoris And Preliminary Application

Some kinds of diseases are related with porcine circovirus type 2 in porcine groups including PMWS, PDNS, CT, et al. Especially, PMWS is the most serious and has made great economic losses in the world. China can not escape by sheer luck. 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 nucleocapsid protein of PCV-2 encoded by ORF2 is a major structural protein of virus and has specific immunogenicity .The Cap 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 Cap protein and protect against the diseases associated with PCV-2.1. we designed one pairs of primers and amplified the ORF2 genes from the vector pcDNA3-0RF which was constructed by Tan, and the PCR products were inserted into secretory p PICZ. A P. pastor is expression vectors. Recombinant strains with ORF2 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 ORF2 protein were: 28-30 ℃, 225rpm, when BMGY with OD600 being 3, the recombinant Picha Pastoris was add to the BMMY medium of pH 7.5which with OD600 being 1.5.Then the solution was incubated 72h and methanol was added to keep its concentration up to 0.5% between 24h.After induction under optimal conditions, the yield of ORF2 protein could be added up to 278μg/mL. It was found that the supernatant should be conserved in -20 ℃ after comparing effects of different conservation temperature on lysis of protein ORF2.3. An indirect ELISA using protein ORF2 as coating antigen was established to monitor antibodies level of pigs against PCV-2 and its operation methods were optimized. The optimal operation methods were: A protein concentration of 42.5μg/mL was coated overnight at 4℃ on 96-well microtiter plates and the plates were subsequently blocked 3h with 1% BSA ,and then incubated for 45 min with sera diluted by 1% BSA, after washing the plates, the anti-swine immunoglobulin conjuate was added and incubated for 30 min at 37℃ or 45min at RT, and then the indicator OPD was added and incubated for 15 min at 37℃ or 20min at RT, 0.12% HF was added before OD was measured at 630nm.200 pieces of sera from pigs infected by PCV-2 were examined by this method and compared with the method of latex agglutination diagnostic kit which constructed by HuaZhong Agriculturaluniversity. The agreement ratio between the two methods was 90%. Experiments suggested that the method we constructed was promising.