Construction Study Of An ΔapxⅠC/ⅡC Mutant Of Actinobacillus Pleuropneumoniae Serovar 5

Pleuropneumoniae Actinobacillus (Actinobacillus pleuropneumoniae, APP) is a herd highly contagious respiratory infectious disease, which can cause porcine contagious pleuropneumonia (Porcine contagious pleuropneumonia, PCP). Therefore, building the immunogen of APP gene attenuated deletion mutant has become an important research direction of new vaccines for preventing the disease. in this study, the deletion ApxⅠC and Apx Ⅱ C double gene mutant SW1ΔⅠC/ΔⅡC was constructed based on single-gene deletion strains SW1ΔⅠ C, and relative research was done to its biological characteristics and immunogenicity.1. Construction of the Actinobacillus pleuropneumonia SW1ΔⅠC/ΔⅡC1.1 Construction of the homologous recombinant vector pBOSKΔⅡCset APP5 SW1 genome as a template, a pair of primers were designed, and amplified the upstream 2654bp and downstream 2039bp sequence TA and cloned into pMD19-T. Detected with PCR, digestion and sequence, connecting to the vector pBS-OSK using SacⅠ, SalⅠ sites by PCR and restriction analysis, then correct, the test successfully constructed APP5 SW1 strain recombinant transfer vector pBOSKAIIC of deleting Apx Ⅱ C gene 451bp.1.2 Selection and construction of double mutant SW1ΔⅠC/ΔⅡCElectroporated the recombinant transfer vector pBOSKAIIC into the App serotype 5 single-gene deletion of strains SW1ΔⅠC, which could improve the homologous recombination between homologous sequence and parental strain genes. Screened the first homologous recombination, having a kanamycin-resistant colonies from the TSA plates containing kanamycin, to obtain two PCR identification respectively A 738 bp fragment of 287bp and around the strip, by resistance experiment proved correct strain transfer a TSA plate containing sucrose, a second homologous recombination, screening containing sucrose resistant colonies were done by PCR to give a 287bp fragment. In this research, a dual deletion mutant deletes Apx ⅡC upon the loss of single gene was successfully constructed, which was named SW1ΔⅠC/ΔⅡC.2 Biological Characteristics and immunogenicity of the mutant SW1ΔⅠC/ΔⅡCGrowth characteristics test confirmed that mutant SW1ΔⅠC/ΔⅡC’s growth is not significant, comparing with the strains, genetic stability tests confirmed that continuous pass 10 generations in vitro the gene still stable genetic and hemolysis test confirmed in vitro is not the performance of hemolytic activity, and cells toxicity test confirmed its cytotoxic was weaker than the parental strain. Determination of the LD50, the mutant mice half lethal dose of 5.0×108CFU, the parent strain of 5.0×106 CFU, a difference of 200 times, toxicity to mice significantly reduce; in immune protection test, mice immunized with the mutant strain of 10 times the LD50 of the parental strain as well as the heterologous serotypes provide 100% protection, and can still provide some protection to 100 times the LD50 of the heterologous serotype; using ELISA immune serum confirmed can induce mice to produce antibodies against APP and the highest antibody concentration in the three priod. These tests show that the virulence of the mutant was significantly reduced, and this mutant can provide a more comprehensive and effective protection for further study of the mutant APP homologous and heterologous serotypes infection.