Construction Of The Gene Deleted Attenuated Mutant Strain ApxIIC~-/apxIN~+ Of Actinobacillus Pleuropneumoniae

Porcine contagious pleuropneumonia(PCP)is an infectious porcine respiratory tract disease causing severe economic losses worldwide in the swine industry,caused by Acrinobacilluspl europneumoniae(APP).Immunization is the main method to prevent and control this disease.The currentcommercial vaccines are still primarily killed whole cell bacteries and subunit vaccines,which generally reduce mortality from APP infection but frequently fail to prevent severer morbidity and economic loss due to the chronic effects of the disease on growth rate and feed efficiency.In contrast,natural and experimental infection can induce protection against any heterogonous sera type.Thus,live attenuated vaccines might be a viable approach to solve this problem.The previously described methods for the construction of mutant strains of A.pleuropneuminiae have some disadvantage.In this research,apxâ…¡C gene in Actinobacillus pleuropneumoniae serovar 7 strain WF83 was replaced by kanamycine resistance gene so that Apxâ…¡could not 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.1 Construction of the Recombinant Transfer Vector pBSLNKARTwo 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 2,200 bp in the 5' region of apxâ…¡C gene in APP-7 strain WF83 as left homologization brach and 1,400 bp 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.2 Selection and Identification of the Gene Deleted Attenuated Strain apxâ…¡C~-/apxâ… N~+ of Actinobacillus pleuropneumoniaeThe linearized recombinant transfer vector pBSLNKAR 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 pBSLNKAR.Experiment of haemolysis confirmed that mutant had no ability of haemolysis.Salty of injected mice experiment confirmed that cytotoxicity of mutant was attenuated significantly so that mutant is safe to mice.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.