Construction Of The Gene Deleted Mutant Strain ApxIIC^-/kan^r+ Of Actinobacillus Pleuropneumoniae

In the research,apxâ…¡C gene in Actinobacillus pleuropneumoniae serovar 7 strain WF83 was replaced by kanamycine resistance gene so that Apxâ…¡couldnot be activated.The mutant strain had not the original virulence of hematocytolysis and cytotoxicity.It was attenuated,which was used as live vaccine to research its immunoprotection for mice to field Actinobacillus pleuropneurnoniae.1 Construction of the Recombinant Transfer Vector pBSKATwo pairs of primers were designed and synthesized on published primers submitted by Prideaux et.al in 1999 and published partial sequence of apxâ…¡operon in Actinobacillus pleuropneumoniae serovar 7(APP-7) strain L25-4 to amplify about 2,200bp in the 5' region of apxâ…¡C gene in APP-7 strain WF83 as left homologization brach and 1,000bp in the 3' region of apxâ…¡C gene in APP-7 strain WF83 as right homologization brach.Left homologization brach was cloned into pBS-T to generate plasmid in which the norientation plasmid was called pBSL.Right homologization brach was cloned into pMD19-T Simple to generate plasmid pMD19-SR.One pair of primers was designed and synthesized on published sequence of pEGFP-N1 to amplify kanamycine resistance gene of pEGFP-N1. Kanamycine resistance gene was cloned into pMD19-T Simple to generate plasmid pMD19-K.Besides,Kanamycine resistance gene generated from digestion of pMD19-K with EcoRâ… and BamHâ… was cloned into pBSL between enzyme sites of EcoRâ… and BamHâ… to generate interim plamid pBSLK.Right homologization brach generated from digestion of pMD19-SR with BamHâ… and Sacâ… was cloned into pBSLK between enzyme sites of BamHâ… and Sacâ… to generate recombinant transfer plamid pBSKA.2 Selection and Identification of the Gene Deleted Attenuated Strain apxâ…¡C^-/kan^r+ of Actinobacillus pleuropneumoniaeThe linearized recombinant transfer vector pBSKA was electroporated into parent strain Actinobacillus Pleuropneumoniae serovar 7(APP-7) strain WF83.Product of the electroporation was plated onto TSB agar containing kanamycine(Kan).After 1-2 days,the recombinant strains were selected.Resistance of kanmycine experiment confirmed that mutant can counteract kanamycine.Dependence of NAD experiment confirmed that mutant need NAD in growth.Identification of PCR confirmed that complete apxâ…¡C gene was substitute with kanamycine resistance gene and there was no presence of pBSKA.Experiment of haemolysis confirmed that mutant had no ability of haemolysis. Cytotoxicity test confirmed that mutant had no cytotoxicity.Safty of injected mice experiment confirmed that cytotoxicity of mutant was attenuated significantly so that mutant is safy to mice.Experiment of genetic stability conformed that kanamycine resistance of mutant is stable in 30 vitro successive generations and I0 vivo generations.All of the above indicated that the gene deleted attenuated strain we constructed was successful,which provided a certain basis for further researching genetic live vaccine with the mutant strain.3 Research of Immunoproteetion of the Gene Deleted Attenuated Strain apxâ…¡C^-/kan^r+ of Actinobacillus pleuropneumoniaeTo evaluate the vaccination efficacy of the gene deleted attenuated mutant strain apxâ…¡C^-/kan^r+,mice were vaccinated with 1.1×10⁸CFU of apxâ…¡C^-/kan^r+ strain via inoculation subcutaneously in the back on day 0 and 14.14 days after secondary vaccination,the immunized mice groups together with TSB control group were challenged with 0.5mL fatal dose of Actinobacillus pleuropneumoniae containing 3.4×10⁷CFU serotype 1,6.8×10⁷CFU serotype 5and 1.1×10⁹CFU serotype 7,respectively.The gene deleted attenuated mutant strain apxâ…¡C^-/kan^r+ offered 100%(10/10) immunoprotection against serovar 1,5 and 7.The result of immunoprotection of mice initially indicated that the gene deleted attenuated mutant strain offered good immunoprotection against both homologous and heterologous serovars of Actinobacillus pleuropneumoniae.The gene deleted attenuated mutant strain apxâ…¡C^-/kan^r+ is a kind of potential APP vaccine strain.