Cloning Of ApxⅠA And ApxⅢA Gene Of Actinobacillus Pleuropneumoniae And Their Expression In Escherichia Coli

Objective: Actinobacillus pleuropneumonie (App) is the causative agent of porcine infectious pleuropneumonia, a highly contagious, fibrinous, hemorrhagic, and necrotizing disease. The virulence of App is multifactorial, but Apx is considered not only the most important virulence factor but also the major protective antigen in App. The protective epitopes might be located at the N-terminal fragment of Apx. The research aimed at producing the gene fragments of apxIA(1149bp) and apxIIIA(1194bp) which coded the N-terminal fragment of ApxI and ApxIII, and expressing them in prokaryotic expression system by molecular biologic technology. It would lay a foundation for developing new effecient subunit vaccine and specific diagnosis test of App in pig. Method: Genes apxI(3069bp) and apxIIIA(3159bp) were amplified by PCR with a pair of specific primers and. genomic DNA of App as template in present study, Amplified fragments were inserted into pMD18-T vector, then transformed into E.coli JM109. The recombinant plasmid pMD-apxIA and pMD-apxIIIA were identified by restriction analysis, PCR and DNA sequencing. Then target genes, that were partial fragments of aPxIA(1149bp) and apxIIIA(1194bp) which coded the N-terminal fragment of Apxl and ApxIII, were amplified by PCR with a pair of specifically expressive primers and the recombinant plasmid as template respectively. The expression vector pET-28a was digested by the same enzymes respectively. The target genes were subcloned into vector pET-28a, then transformed into E.coliDH5a. Recombinant expression vectors pET-apxIA and pET-apxIIIA were transformed into E.coli JM109(DE3) for expression induced by IPTG. The bacteria containing pET-apxIA, and pET-apxIIIA were collected at different time and subsequently were examined by SDS-PAGE and Western-blotting.Dissolved bacteria, the inclusion body was purified and washed with low density urea, and then the purified products were denatured with high density urea. Results: It was proved that the acquired recombinant plasmid pMD-apxIA and pMD-apxIIIA contained complete apxIA(3069bp) and apxIIIA(3159bp). The homologies of the nucleotide sequence of apxlA were 93% and 99.2% comparing with that of strains AF363361 and D16582, and of flpxIIIA were 96% and 99.7% comparing with that of strains EWSDSL12145 and X68815 respectively. The target genes coded the N-terminal fragment of ApxlA and ApxIIIA wereinserted into expression vector pET-28a(+). Recombinant expression vector pET-apxIA and pET-apxIIIA were obtained successfully. Results of SDS-PAGE and Western-blotting showed that the bacteria containing pET-apxlA and pET-apxIIIA could express successfully in E.coli JM109(DE3). Quantity of both the target proteins reached up 30% in the total bacteria protein, and their molecular weights were 41ku. The fusion proteins could be recognized by the positive serum from Apx-immunized swine. It was proved that the expressed proteins were insoluble and existed in the form of inclusion body. Primary purified protein was obtained.