Research On Prokaryotic Expression And Immuncompetence Of Type â…¢ Fimbriae Adherence Gene From A Fowl Klebsiella Pneumoniae

Fimbrial is one of important causative agent factors in Klebsiella Pneumoniae(Kp).It is very important for its diagnosis and prevention to research Kp pathopoiesis principle. mrkA and mrkD are typeⅢfimbriae Adherence gene which are the capital reasion leading to Kp causative agent.Either birds or swine and people are both to be infected with Kp. The infection mortality rate may reach as high as 60%. Because its drug resistance gradually strengthens,more and more topics which people pay attention are about the treatment and disease prevention of Kp.In this studay , Virus of the local isolate Kp5,Kp6,Kp7,Kp8,Kp9 were propagated in bouillon culture-medium(MH) .They are all subsp.pneumoniae which were identified by molecular biology and bacteria morphous. According to the reported mrkA and mrkD nucleotide sequences in GenBank.,two pair of primers were designed to amplify A and D gene with Oligo 6.0 and primer 5.0 software. The A and D gene were cloned into pMD18-T simple vector and sequenced. The constructed recombinant plasmid was analyzed by PCR,rest riction enzyme digestion and sequence analysis. Results showed A and D gene respectively shares 87%～100%,80%～100%.homology with that from Genbank. They were settled the foundation to further study structure and biological funtion of Kp by obtaining A gene and D gene .The target gene and expression vetor pGEX-4T-1 were digested with EcoRI and BamHI respectively. The target gene were subcloned into vetor pGEX-4T-1 and named as GST-A and GST-D with intertest gene which were identified by restriction analysis,PCR and DNA Sequencing. Then the recombinant was transformed into E.coil BL21(DE3) for A and D expression.The insert gene was induced to express in E.coil with IPTG.The bacteria containing GST-A和GST-D were collected and examined by SDS-PAGE.Results showed that the GST-A和GST-D gene of KPN could express successfully in E.coil. Molecular weight of the fusion protein was 45 KDa. and 61 KDa which contained GST tag(26 KDa) .The fusion protein could be recognized by the positive serum of KPN .We obtained the typeⅢfimbriae adherence gene sequence of Kp for the first time at molecular level in our country ., and t hen expressed A gene and D gene in prokaryotic system..Our research will provide the certain rationale for manufacture the genetic engineering vaccine and the reorganization vaccine to prevent Klebsiella Pneumoniae disease.