Construction And Immunogenicity Of An â–³apxIC/ompP2 Mutant Of Actinobacillus Pleuropneumoniae Serovar 5 Gomphosised OmpP2 Gene Of Haemophilus Parasuis Serovar 5

Actinobacillus pleuropneumoniae (APP) is a pathogen of pig contagious respiratory disease. It can cause porcine contagious pleuropneumonia (PCP). Haemophilus parasuis (Hps) is a kind of normal flora, which is cloned on respiratory tract in pig. It has been confirmed that Hps could cause occurrence of Hps disease in pigs as an opportunistic pathogen. Both of the two pathgens can cause huge economic losses in the pig industry. In the current, the attenuated deletion mutant of bacterial vaccines against infectious diseases became an important research direction. In this study, A. pleuropneumoniae serovar 5 SC-1 strain was used to construct an A. pleuropneumoniae ApxIC gene deletion mutant namedΔapxIC/ompP2, which gomphosised ompP2 gene of H. parasuis serovar 5 HS80 strain. We also studied their biological characteristics and immunogenicity.1. Construction of the recombinant transfer vector pBOSKΔIC/ompP2Construction of the middle recombinant transfer vector pBOSKΔIC-1:1434bp downstreams sequences of ApxIC gene was amplified from the APP SC-1 strain. It was the right homologous arms, and added sites Spel and Mlul in its upstreams. The amplifition fragment was TA-cloned into pMD19-T Simple, we got a 1400bp fragment by PCR, and it was verified to be a correct one after restriction enzyme digestion. Then it was used to replace the origined right homologous arm of pBOSKΔIC, and got a fragment of about 1400bp. Subsequently, about 1400bp and 7000bp fragments were obtained by identifition of restriction enzyme digestion. This study inferred that the vector pBOSKΔIC that inserted the MluI restriction site into its right homologous arm constructed the middle recombinant transfer vector pBOSKΔIC-1 successfully.Construction of the recombinant transfer vector pBOSKΔIC/ompP2:Amplified 1107bp fragment of ompP2 gene was amplified from the Hps HS80 strainAfter TA-cloning the amplified fragment into the pMD19-T Simple, we got a about 1100bp fragment by PCR, which was correct by identifition of restriction enzyme digestion. Then we connected the fragment to the recombinant transfer vector pBOSKAIC-1 between the Mlul and Spel sites, and got a fragment of about 1 lOObp by PCR. About 11OObp and 8500bp fragments were obtained by identifition of restriction enzyme digestion. This study constructed recombinant transfer vector pBOSKAIC/ompP2 successfully, which inserted Hps ompP2 gene into the middle recombinant transfer vector pBOSKAIC-1.2. Construction of the gene deletion mutantΔapxIC/ompP2The recombinant transfer vector pBOSKAIC/ompP2 was electroporation into APP SC-1 competent for the first homologous recombination, and chose the kanamycin resistance colonies from the TSA plate containing kanamycin. About 1400 and 800 bp fragments were obtained by PCR. Transferred the correct strains to the TSA plate containing sucrose for the second homologous recombination, and choose the sucrose resistant colonies identified by PCR. This study screened a mutant of A. pleuropneumoniae serovar 5, namedΔapxIC/ompP2, which gomphosised ompP2 gene of H. parasuis serovar 5.3. The biological properties and immunogenicity of the gene deletion mutantΔapxIC/ompP2The resultsshowed the growth characteristics of the mutantΔapxIC/ompP2 and the parent strain were no significant differences; the genetic test confirmed that the mutantΔapxIC/ompP2 in vitro stability of a continuous gene transfer still stable after 10 generations of genetic; the hemolytic activity test confirmed that the hemolytic activity of the mutantΔapxlC/ompP2 did not show hemolytic activity in vitro; the cytotoxicity test confirmed that the cells of the mutantΔapxIC/ompP2 were less toxic than the parent strain; the median lethal dose of the mutantΔapxIC/ompP2 was 1.0×107CFU, and the parent strain was 3.5x105CFU, the difference of 30 times, and reduced the toxicity in mice significantly. In the immune protection experiments, mice immunized mutantΔapxIC/ompP2 could provide 100%protection to 20 times the LD50 of the parental strain, and provide 62.5%protection to 5 times the LD50 of the Hps HS80; immune serum by ELISA test confirmed that the mutantΔapxIC/ompP2 could induce mice antibodies against APP and Hps, and the concentration of antibody in the third immunement was the highest. These tests showed that this mutant reduced virulence significantly, it can provide mice of APP infection more comprehensive, and provide effective protection of Hps infection at the same time. It was a basis for further study of genetic engineering and the foundation for practical applications.