Cloning,Expression of apxl Gene of Actinobacillus pleuropneumoniae and Development of ELISA

Based on the published nucleotide sequence of the apxICA of Actinobacillus pleuropneumoniae in Genbank(S4074),a pair of primers were designed.A 3640bp(4687-8326bp)gene fragment was amplified by PCR from the isolated strain of A.pleuropneumoniae serover 1.Then,it was cloned into pMD18-T,identified by both restriction endonuclease and sequence analysis,and inserted into pET-28a expression vector to yield the expression plasmid.SDS-PAGE result indicated expression of apxICA in BL21(DE3),Western blot analysis showed the protein's immunogenicity.Using the expressed,ELISA was established to detect serum antibody against ApxI.The feature of ELISA to detect highly virulent A.pleuropneumoniae strains infection was proved by primary clinical application.