Swine Infectious Pleuropneumoniae The Bacillus Apxic Gene Deletion Mutant Construct

Actinobacillus pleuropneumoniae (APP) is the etiological agent of Porcine Contagious pleuropneumonia (PCP),a severe respiratory contagious disease causing huge economic losses in the pig-raising industry. At present, vaccination is one of the main measures to prevent and control the disease. However, complete bacterium killed and subunit vaccine that use on production cannot reduce the disease incidence rate and provide complete cross protection to the heterologous serotype infection. Natural infection of APP can induce animal organism to elicit protective antibodies for heterologous serotype, so the research of attenuated vaccine is the important research orientation of studying APP vaccine. In the study, ApxIC gene deletion mutant of Porcine Actinobacillus pleuropneumoniae was constructed.1.Construction of APP recombinant pBOSKΔICConstruction of APP plus-minus screening expression cassette:Amplified omlA gene promoter, levansucrase gene and kanamycine resistant gene (Kanr) from APP5,Bacillus subtilis and pEGFP-N1 by PCR, and the gene sizes were 266bp,1422bp,and 795bp respectively.Which were TA cloned into pMD19-T, then the vectors were identified exactly by PCR, enzyme digestion analysis and DNA sequencing. Arrange by order, omlaP,sacB,Kanr were digested and inserted into the pBluescript II SK+vector between the site of Xhol and KpnI,to create a recombinant vector pBS-OSK. The three inserted gene combined to be the plus-minus screening expression cassette. The E.coli including pBS-OSK had kanamycin resistance and sucrose sensibility. The results showed that plus-minus screening system on the basis of Kanr and sacB was constructed successfully.Construction of APP recombinant pBOSKΔIC:Taking APP5 K17 strain as a template, amplified the sequence of upstream 2895bp and downstream 1434bp to be left and right homologous arm respectively. The fragments were TA Cloned into pMD19-T; then the recombinant were identified exactly by PCR, enzyme digestion analysis and DNA sequencing, and digested and inserted into the pBS-OSK vector between the site of SacⅠand SaiⅠto construct pBOSKΔIC deletion of 457bp ApxⅠC,which was identified in a correct way.2.Construction of APP ApxIC gene deletion mutants By electrotransformation, the recombinant pBOSKAIC was transformed into APP5 K17 parent strain. Cultured it in TSB culture medium,3 hours later, then coated bacteria on the kan TSA plate and cultured 18h. The Kanr colonies were inoculated into 10% sucrose TSA plate to identify the sucrose sensibility, which was tested by PCR.Picked out a random colony identified exactly, that was inoculated into TSB culture medium for the night in order to stimulate the second homologous recombination. Diluted cultures was coated on the 10% sucrose TSA plate without kan,then the screened colonies with sucrose resistibility were coated on TSA-10%Sucrose-Kan plate.Identified the colony with kan sensitivity and sucrose resistibility by PCR, and the correct ApxIC gene deletion mutants(K17ΔICwas obtained. The research showed that,K17ΔIC had a good genetic stability in vitro after 10 generations by transfer of culture experiment, completely lost cytotoxicity, and the virulence reduced significantly which was safe to mice.It demonstrated that the gene deletion mutant was constructed successfully. All of which establish the important base for the further research of live attenuated genetic engineering vaccine.