Isolation And Identfication Of Actinobcillus Pleuropneumoniae And Its ApxⅠA And TbpB Gene Cloning And Prokaryotic Expression

Outbreak Actinobcillus pleuropneumoniae from the pig in Sichuan,a strain bacterium was isolated from that farm,which was identified as serotype 1 of Actinobcillus pleuropneumoniae by morphological,serological and molecular biology tests.The antibiotic sensitivity test implied with it was sensitive with the tetracycline,Ceftriaxone, Norfloxacin,Trimethoprim+Sulfamethoxazole,ciprofloxacin,and drug resistance with the Kanamycin,Amikacin,Cefalexin,Vancomycin,Ampicillin,Gentamicin.The PCR identification refer to foreign report.According to the conservative weak haemolytic activity gene sequences(APXâ…£A) of Actinobcillus pleuropneumoniae,we designed the primers and identified the strain by PCR.According to the seqence of the Apxâ… A(X68595) and TbpB(U16019) gene of Actinobacillus pleuropleumoniae(serotype 1) in GenBank,two pair of the specific primers were designed and synthesized,templated by the genomic DNA samples from the stain of APP-CYY.The PCR fragment of Apxâ… A and TbpB gene were cloned about 3089bp and 736bp.The two amplified fragment was cloned into pMD18-T vector and then sequenced analysis.The result indicates that the cloned gene was a complete ORF Apxâ… A and TbpB gene.Sequence comparison between the different serotype1,5,9,10 Apxâ… A gene of Actinobcillus pleuropneumoniae that homology are between 96.8-98.8%.Sequence comparison between two serotype 1 TbpB gene of Actinobcillus pleuropneumoniae that homology are 99.7%and 99.6%.According to enzyme digested the recombinant plasmid of pMD18-T-Apxâ… A and pMD 18-T-TbpB,the fragment of Apxâ… A and TbpB gene got,and the fragment inserted the prokaryotic expression vector of pET32a(+),constructing the expression plasmid of pET32a(+)-Apxâ… A and pET32a(+)-TbpB.The positive recombinant plasmid was transformed into E.coli BL21,and was induced to express the protein of Apxâ… A and TbpB.A fusion protein about 125KD and 44KD was identified by SDS-PAGE, Western-blot identify positive.