Construction And Application Of Selective Cloning Vector For Genes Encoding Exported Proteins Of P. Multicida

Based on the finished vector pMBL, a 260bp fragment of gene encoding Lac promoter was amplified from pUC18 vector, and then inserted into pMBL to construct the new selective cloning vector pMB-Lac, which was 3.72kb in length. Meanwhile, another 4.74kb cloning vector pMB-Ara with inducible Ara promoter for genes encoding exported proteins was obtained. Both results of the restriction enzyme digestion and sequencing demonstrated the correctness of the constructed vectors.The Tet resistance gene which encodes a transmembrane protein, was applied to verify the effectiveness of the reporter in the two vectors. TheΔP Tet gene (i.e. Tet gene without promoter) was amplified from pBR322 vector and ligated to the two vector which had been cut with either enzymes of EcoR I and BamR I, or EcoR I and Bgl II, or EcoR I and Bcl I. Kan and Amp double resistant colonies only grew with the EcoR I and BamR I combination. Restriction enzyme digestion and sequencing results of the recombinant plasmid showed that Lac/Ara promoter in the vector promoted the co-expression and transmembrane secretion of downstream report gene andΔP Tet.To evaluate the influence of promoter in the vector frame on leaking expression of cloning genes,ΔP Tet gene was cloned into pMBL, pMBLΔPTet plasmid was constructed for comparison. The transformant grew on Kan plate, but little on Amp+Kan plate whenever induced or not. To analysis and compare the extent of regulation range,leaking expression and the best induced condition of Lac promoter and Ara promoter, the transformants of pMB-LacΔPTet and pMB-AraΔPTet vector were induced under varied conditions including different inducers and concentration. To eliminate the possibility of destroying SD sequence when removing the promoter from Tet gene, pMB-LacΔPT7-Tet plasmid was construted and then was compared with pMB-LacΔPTet. After transformed, both of them grew on Amp16,Amp32 and Amp64 plate in a certain extent when induced with 1.5Mm IPTG, the transformant carrying T7-SD grew much better than that containing Tet-SD counterpart in comparison. Bacterial carrying pMB-AraΔPTet plasmid grew well on Amp16, Amp32,Amp64 and Ampl28 plate when induced by 0.5% arabinose, but much slower than those on Kan plate; after induced by 0.3-10% arabinose, we found that when induced by 1.25% arabinose, most colones was achieved on Amp50 plate. Analysis of the stability of the new constructed recombinant plasmid showed that the loss of vector was not obvious and the vector frame was stable after 10 and 20 generations.pMB-Ara vector was selected to clone genes encoding exported proteins with constant (structural) or regulatory expression mode of P. multocida C48-19. The genomic DNA was randomly digested with Sau3A I and was then ligated to pMB-Ara vector. Clones grown on Amp+Kan plate was labled and hybridized with those on Amp+Kan+l%arabinose plate. Five clones which only grew on induced plate was obtained(screened out), and named 62~#,88~#,89~#,107~#and 108~#. From the analysis of inducible activity and growth traits, we found that 88~# clone could only grow on Amp+Kan plate with 1.25%arabinose. It was conclude that the detected Amp activity of 88~# clone was promoted by Ara promoter in pMB-Ara vector rather than by 88~# clone. However, 19~# clone showed the trait of constant expression mode because of its growing on the Amp+Kan plate with or without inducer.Sequencing,homology analysis and SignalP results of 88~# clone demonstrated that the complete promoter structure located in the upstream of starting codon and it was highly homology with artl gene of Pm70. The gene inserted into 88~# clone was predicted to encode exported proteins with regulatory expression mode. Whereas 19~# clone corresponded to unknown gene of Pm70 and was belonged to constant expression mode. In conclusion, the vectors could be used to selectively clone genes encoding exported proteins with constant and regulatory expression mode.This research was a fundamental research for further analysis of genes encoding cross protection factors, immunogens and virulence factors of P. multocida.