The Apx Ⅱ A Gene Of Actinobacillus Pleuropneumoniae Prokaryotic Expression And Research In Immunogenicity

The Porcine Contagious Pleuropneumonia (PCP) is composed of Actinobacillus pleuropneumoniae (APP), caused a highly contagious disease, the development of the disease threatening the world pig industry, bring huge economic losses to the pig industry. Research on APP virulence factors is the focus and difficulty of this disease research.1. construction of prokaryotic expression vector pET-ApxⅡAThis study successfully amplified APP serum encoding toxin protein1gene of Apx Ⅱ A gene by PCR method, the size of this gene is2871bp, the gene sequence was cloned into the prokaryotic expression vector pET-28a, obtained the prokaryotic expression vector pET-Apx Ⅱ, and identified by restriction enzyme digestion and sequencing. The results show that, to construct the prokaryotic expression vector of pET-Apx Ⅱ Apx Ⅱ A gene.2. The APX ⅡA gene expression product immunogenicityTo construct the prokaryotic expression vector pET-Apx was transformed to BL21(DE3) competent cells, induced expression, The recombinant plasmid in E.coli OD600reached0.8, add IPTG to a final concentration of1.0mmol/L, keep good expression after3H incubation, the size of the expression product was about110KDa, And the use of SDS-PAGE and Western-blot were detected, confirmed that the expression product had good immunogenicity. The expressed protein was purified after immunization of mice. A week after each immunization, detection of antibody by toxin Ⅱ established ELISA, Three free, found that the antibody titer increased significantly, confirmed the expression product has good immunogenicity.3. The APX ⅡA protein protective efficacy in mice100mice were randomly divided into5groups, the serum type1inactivated whole cell vaccine group, serum type7inactivated whole cell vaccine group, Apx Ⅱ A protein group, serum type7inactivated vaccine+Apx Ⅱ A protein group and PBS control group. A week after each immunization, detection of antibody by toxin Ⅱ established ELISA, third a week after immunization, with concentrations were1×106CUF/ml and1.5×106CUF/ml type1and type7strain of virus attack. The results show:Mice were given a single Apx ⅡA protein immune protection against serotype1attack rate was60%, protection of serotype7was 50%, have protective immunity to the homologous serotype and heterologous serotype;And the serum type7Protection of inactivated vaccine+Apx II A protein group was70%and90%respectively, The results show that, Apx II A protein can enhance the immune effect of inactivated vaccine in a certain extent.