Study On Irp1 And Irp2 Genes Of HPI In Escherichia Coil

High-pathogenicity Island (HPI) is present in pathogenic yersinia. This 35 to 45 kb island carries genes involved in synthesis, regulation and transport of the siderophore yersiniabactin. The study indicated, the HPI appears to be widespread in various enterobactia strains, for example, E.coli strains, Salmonella and Klebsiella, etc. HPI may be distributed in various species through horizontal gene transfer. HPI contains a core region composed of 11 genes, irpl and irp2 are iron regulatory genes encode two iron-repressible high-molecular mass proteins designated HMWP1 and HMWP2, which are involved in the ability of iron intake, and expressed only in pathogenic bacteria. HPI is not only attributed to the pathogenicity of bacteria and specific phases of the infection progress but also plays an important role in the evolution of bacterium.In this research the HPI-positive E. coli is isolated from Jiangsu area. Two pairs of primer were designed according to the published gene sequences of irpl and irp2 for PCR amplification and got the target fragments. The PCR products were subcloned into the pUCm-T vector obtained recombinant clones pUCm-irpl and pUCm-irp2, which were analyzed through sequence then connected to the expression vector pGEX-6p-1, the recombinant plasmids were renamed as pGEX-irpl and pGEX-irp2, transformed into E.coli BL21(DE3) competent cells.The recombinant strains were induced by the addition of IPTG at 37℃, the lysate of recombinant strains was analyzed through SDS-PAGE, proving that recombinant strains which contain recombinant plasmid can respectively express 41 KDa and 40 KDa fusion proteins, which were renamed as GST-HMWP1 and GST-HMWP2. The purity of soluble protein achieves 95% by GSH-Sefinose purification. The New Zealand rabbits were immunized with purified protein, producing the polyclonal antibody, and it facilitates us to study the immunologic functions of these proteins.For further research of the relationship between irp1, irp2 genes and the virulence of HPI, in this study, Red/ET recombination was brought in to knock out irpl and irp2 genes. Two pairs of primers were designed and synthetized according to the published sequences of irp1, irp2 and FRT-PGK-gb2-neo-FRT genes to amplify functional cassette flanked with homology arms. In the first electroporation, E.coil strains of S793103 and S711100 which would be modified, was transformed with the expression plasmid pRedET, incubated at 30℃; the expression of genes mediating pRedET was induced by the addition of L-arabinose. After induction, the cells were prepared for electroporation and the PCR product carrying the homology arms was electroporated. Red/ET recombination inserts the functional cassette into the target locus to produce homologous recombination. The control test is necessary in all process.