Establishment Of PCR For Detection Of Actinobacillus Pleuropneumoniae And Expression Of It's Outer Membrane Lipoprotein Gene

Actinobacillus pleuropneumoniae (APP) is an important pathogen causing Porcine contagious pleuropneumonia,which causes enormous economic losses world-wide. Previous studies have shown that it's immunogenicity is determined by the Outer Membrane Lipoprotein(OML)of APP to a large extent.A pair of primers was designed based on the published OML gene sequences of APP. After amplified from QH-1 and HN-7 strains by polymerase chain reaction(PCR),the gene was cloned into the pMD18-T vector to construct the recombinant plasmids AppOml-1 and AppOml-7. By sequencing and analizing the OML genes of the recombinant plansmids AppOml-1 and AppOml-7 with DNAstar, it were indicated that the nucleotide sequence of the OML gene from QH-1 strain exhibited over 99.1% homology with reference serotypes 1,9,11 and 12, the nucleotide sequence of the OML gene from HN-7 strain exhibited over 97.3% homology with reference serotypes3,4,6 and 7, and they also exhibited much lower homology than other reference strains.The analysis also showed that there were conservative segments in both ends of the OML genes from QH-1 strain and HN-7 strain. According to this novel discovery,a pair of specific primers was designed and with the primers,a 980bp DNA fragment could be amplified from all standard App strains(from 1 to 10). On the contrary,some pathogenic bacteria isolated from swine,which can cause the similar clinic symptom,had the negative results. The lowest concentration of testing DNA was 2pg/ml. To verify this method of PCR for quick detection,6 strains of App from clinic were amplified,and all results were positive,they were coincided with bacteriological determination. These experimental results demonstrated that it could be used as a quick-detection method for APP. The segment containing complete open reading frame(ORF) of OML gene was amplified from the recombinant plasmid AppOml-1 by PCR. After digested with EcoRâ… / Pstâ… , the gene was subcloned into prokaryotic expression vector pPRoEX HTb and transformed into E.coli BL21. The exactness of ORF of positive clone was confirmed by sequencing. The recombinant expression plasmid pPRocAppOml-1 was constructed. Subsequently, the transformant was induced by IPTG. The bacteria was collected every two hours and analyzed by means of SDS-PAGE and Western blot. The results showed that the cAppOml-1 gene of QH-1 was successfully expressed in E.coli BL21 and the product, a kind of fusional protein,was approximately 45ku in size, it accounted for 25% of total protein. The product could be recognized by positive serum of APP.