Molecular Detection Of Actinobacillus Pleuropneumonia Infection

According to the published genome sequences of Actinobacillus pleuropneumonia (APP) on GenBank,contain serotype 1 and serotype 3.seven pairs of primers were designed and the Apxâ…£A gene of serotype 5 APP was amplified by PCR and cloned by PMD-18T.The sequenced results shows that the Apxâ…£A gene of serotype 5 APP was 5586 bp in length.The nucleotide sequence homology of Apxâ…£A gene of serotype 5 APP with those of serotype 1 and serotype 3 in GeneBank were 95.5%and 95.4%respectively. The amino acids homology of APP serotype 5 gene with those of published in GeneBank was 98.1%.Bioinformatics analysis of this sequence shows that APXâ…£A protein was hydrophilic and highly antigenic,possessing a obvius transmembrane region and 55 antigenic determinants.Analysis of the secondary structure of amino acid sequence,hydrophobicity,antigenic determinant,and transmembrane region of APP serotype 5 Apxâ…£A gene,select a strong antigenic region N,express it in prokaryotic cells.The Apxâ…£A gene was obtained by PCR with the APP serotype 5 genome as template and cloned into PMD-18T vector.It was indenitified by PCR,restriction endonuclease analysis,DNA sequencing and then subcloned into EcoRâ… /Xhoâ… site of prokaryotic expression vector PET-32(+) and recombinant expression plasmid PET32atApxâ…£A was constructed successfully.the plasmid was transformed into Eschercha coil BL21 and 62KDApxâ…£A recombinant protein was expression with IPTG inducer.The expression product were mainly non-soluble inclusion body.The expression conditions were determind by optimizing induction temperature,induction time and concentration of IPTG.An indirect ELISA method detecting APP infection was estabished based on APP recombinant protein as coating antigen.It is positive when the OD₄₅₀≥0.31,and it negative when OD₄₅₀<0.31.The result showed that the method can differentiate APP infection and other infections,such as porcine reproductive infection and respiratory syndrome infection,swine streptococcosis infection,swine fever infection and swine pasteurellosis infection. The method can be applied in diagnosis of and detection APRA pair of primers were designed according to the sequence of the a Apxâ…£A gene,And a nucleic method of detecting APP was estabished.Specificity of the PCRs showed that the test got the negative results by these PCRs with Actinobacillus pleuropneumonia related Pasteurella multocida,streptococosis,colibacilusis,Salmonellosis and Cyanomycosis.The sensitive of PCRs is 2500 bacteria.At the same time,the APP infected model was estanblished on mice,After the organize was checked,it showed that the organize couldn't amplified specificity strip by directly checking out inspected tissue or tissues of heart,liver,spleen,lung and kidney cultivated 2h;after cultivating 4h of all tissues,they amplified weak strip only in liver and kidney,after cultivating 6h of all tissues,heart,they amplified specificity srip including liver,spleen,lung and kidney.