Expression And Monoclonal Antibody Preparation Of The P30Recombinant Protein Of African Swine Fever Virus

African swine fever (ASF) is a highly lethal disease in pigs caused by African swine fever virus (ASFV). The mortality of the virulent strain infection can be100%. The disease has being prevalent in many African countries and has transmitted recently to our neighboring countries including Georgia and Russia, which poses a great threat to swine industry in China. ASFV can use African wild pigs and soft ticks as the virus repertoire and transmission vectors with highly complicated infection and immune evasion mechanisms. Precently, no vaccine is available and thus quick accurate diagnosis is very important for prevention of transmission and control of the disease.P30is one of the major structural and highly immunogenic protein of ASFV, which has been used as the antigen for diagnosis of the virus. In this study, we optimized the p30gene sequence of E75strain ASFV for E.coli expression and cloned the synthetic sequence into prokaryotic expression vector pET-30a. The recombinant vector pET-P30was transformed into BL21(DE3) E.coli and expression of the His-p30fusion protein was induced by IPTG. SDS-PAGE analysis showed that an expected30-kDa recombinant was expressed mainly as inclusion bodies.By using affinity chromatography, a highly pure His-p30fusion protein was obtained and used to immunize BALB/c mice. After immunization for4times, the mouse serum was collected and the antibody titer up to1:1638400was detected by indirect ELISA using His-p30as the antigen. The spleen cells of the immunized mice were fused with SP2/0myeloma cells and the positive cell clones were screened by ELISA using His-p30as the antigen. After3cycles of subcloning, Two positive cell lines were obtained. Indirect immunofluorescence assay showed the monoclonal antibody in the cell supernatant reacted with p30protein expressed in Marc-145and Western blotting showed that the monoclonal antibody in the cell supernatant reated positively with His-p30fusion protein, but not with the His tag. The two hybridoma cell lines were injected peritoneally into mice and monoclonal antibody titer up to217was detected in the ascites.