Cloning And Expression Of ApxIVA Specific Fragment Of Actinobacillus Pleuropneumoniae And Development Of An Indirect ApxIVA3-ELISA

The pleuropneumonia caused by Actinobacillus pleuropneumoniae (APP) is one of the high-contacted and contagious bacterial deseases of the swine respiratory tract, occurring widely in the pig-producing countries around the world. The massive investigation confirmed that Apx is not only the most important virulence factor but also the major protective antigen in APP.Only ApxIV is been secreted by all of the A. pleuropneumoniae serotypes .The research aimed at producing the gene fragments of ApxIVA (440bp) which coded the 3'-terminal fragment of ApxIV, and expressing them in prokaryotic expression system by molecular biologic technology. Developing an indirect ApxIVA-ELISA.It would lay a foundation for developing specific diagnosis test of APP in pig.The gene encoding for protein ApxIVA specific fragment was amplified from APP Hpn-405 chromosomal DNA by using PCR technique. PCR product was cloned, and the strain containing the vectors were selected on LB-plus ampicillin (100μg/ml)plates and digested with enzymes.The ApxIVA genes were sequenced.Comparisons of the ApxIVA amino acid sequence of GeneBank ApxIV sequences showed that them resemble was 99.8%.Plasmids containing the right insertion were sequenced to confirm its identity and retransformed the combination into E.coli.BL21 (DE3) strain. Bacterial lyses prepared from 0.3mmol/L IPTG induced cultures were loaded directly on to SDS-PAGE. Upon induction, the recombinant pET-ApxIVA3 produced indeed a new protein with an APParent MW of 18KDa.pET- ApxIVA 3 was tagged with six histidine residuces at its C terminus and was purified by nicked chelation chromatography. The fusion protein was immunogenic when detected by Western-blot analysis. The indirect ApxIVA3-ELISA is developing .The purified protein after denaturation, renaturation and purification, was used as antigen. The serum of A. pleuropneumoniae serotype 8 infected pigs was used as antibody.The reserch proved that the purified protein detected in A. pleuropneumoniae serotype 8 infected pigs, but not detected in the inactivated APP bacteria vaccinated pigs and healthy pigs. These results suggested that purified protein has good specificity and bioactivity.It can be used not only to detect all serotypes of APP, but also to differentiate the naturally infected and inactivated vaccine immunized pigs.