Prokaryotic Expression Of VP73 Gene And Major Epitope Domain Of African Swine Fever Virus VP73 Gene For Establishment Of Elisa For Detecting African Swine Fever

African swine fever is an acute infectious disease of pigs. It often results in major economic losses to the aquaculture industry because of the high rates of mortality. Due to the complexity of the protective immune response has thus impaired the development of an effective vaccine to prevent the disease. In order to prevent African swine fever virus into China, we reduce infection and transmission ways of the disease as far as possible according to the etiology and epidemiology characteristics of the virus. And application of serology and molecular biology of non-infectious diagnostic method can detect the virus rapidly and sensitively. Thus, the risk of the disease into our country will be decreased to a minimum. We have carried out the following research in order to establish the appropriate diagnostic methods for our country.1 Cloning of African swine fever virus VP73 Gene and its expression in Escherichia coli BL21 (DE3)Recombinant plasmid pPICZαA-VP73 containing VP73 gene and vector pET-32a were digested with EcoRI and NotI. The VP73 gene was ligated into the expression vector pET-32a(pET32a-VP73 ). Recombinant plasmid that was transformed into BL21 (DE3) was highly expressed after induction by IPTG and the VP73 protein was expressed in the form of inclusion bodies. Western blotting analysis revealed that the recombinant protein could react with African swine fever viru positive serum. Its molecular size is about 66KD.2 Epitope prediction of African swine fever virus VP73 gene and Prokaryotic expression of major epitope domain of African swine fever virus VP73 geneAfrican swine fever virus VP73 gene B-cell epitopes were predicted by bioinformatics methods. According to the general forecasting results, major epitope domain of ASFV VP73 gene was amplificated by using PCR and integrated into the Prokaryotic expression vector pET-32a. Recombinant plasmid was named pET32a-VP73L. Recombinant plasmid that was transformed into BL21 (DE3) was induced by IPTG, SDS-PAGE and Western blotting analysis confirmed that pET32a-VP73L expressed and molecular size is about 32KD. 3 Purification of expressed protein and preparation of polyclonal antibodyExpression product of pET32a-VP73 and pET32a-VP73L are inclusion bodies and soluble form respectively. Different methods were used to purify fusion protein. Purified protein of pET32a-VP73 was used to immune a New Zealand white rabbit(1mg/kg).Titer was tested after the third immunization.4 Establishment of indirect ELISA with the expressed proteinTo establish an indirect ELISA method for detecting ASFV antibodies, purified pET32a-VP73L protein was used as the coating antigen and the reaction conditions were optimized as follows:coating antigen for 37℃1h and 4℃overnight at a concentration of 2.5μg/ml. Serum sample(1:10000)and HRP labeled goat anti rabbit IgG(1:5000) being incubated at 37℃1h. The best coated liquid and blocking liquid is buffer bicarbonate(pH9.6)and PBST-1%BSA. Repeated test, crossing test and stability test show that the ELISA assay have a good repeatability, specificity and high sensitivity.