The Construction Of EHEC O157:H7 Mutant Strain, Cloning And Expression Of New Predicted TTSS Molecular Chaperone Genes

Enterohaemorrhagic E. coli (EHEC) is an enteric pathogen associated with diarrhea haemorrhagic colitis and less commonly with thrombotic thrombocytopenic purpura and haemolytic ureminc syndrome[1,2]. mainly, it causes the pathogenesis through TTSS by diversing bacterial virulent proteins into the host cell.At present, five main effecter proteins in EHEC O157:H7 have identified, much fewer than other pathogens, and the functioning of these five could not explain the complex pathogenic mechanism caused by EHEC O157:H7. For an in-depth study of the pathogenic mechanism of EHEC O157:H7, firstly, all the TTSS pathogen genome sequence data have been extracted from the pathogen genome database, then through the modeling analysis, homology analysis and gene mapping analysis, four possible new molecular chaperones of EHEC O157:H7 were predicted. we intended to work on the identification of the categories, their structures and functions of these four new molecular chaperones.λRed in vivo recombination is a new kind of genetic engineering technique based on homologous recombination, allowing variety of DNA cloning and modification on target chromosome. Comparing with the conventional gene engineering techniques, it does not rely on the presence of suitable restriction sites and can be used to insert, delete, combine or substitute DNA fragment at any required position on a target molecule.This research paper presents theλRed in vivo recombination technology. The main technology route is as follows: first we synthesized linear exogenous DNA PCR products by using primers with 40-to 50-nt extensions that are homologous to regions adjacent to the gene which is to be inactivated and template plasmids carrying antibiotic resistance genes that are flanked by FRT (FLP recognition target) sites. The PCR products were electro-transferred into EHEC O157:H7, which has acquired pKD46 and induced by L-arabinose. With the help of the pKD46 Red recombinant system, those new molecular chaperone genes of EHEC O157:H7 were in vivo replaced by linear exogenous DNA. Due to FRT sites flanking kanamycin gene, the latter was eliminated by FLP-promoted recombination events. Using this method, we successfully knock-out 2 of those 4 new forecast molecular chaperone genes,and the recombinants which has realized precisely knock-out were confirmed by PCR identification, DNA sequencing.After the construction of the mutants of EHEC O157:H7, for which ecs4553 or ecs4563 gene was accurately deleted byλRed in vivo recombination technology. We cultured the wild and the mutants of EHEC O157:H7 simultaneity and had found the different secretion protein through the SDS polyacrylamide gelatin electricity which examines their difference of the secretion protein. Then we did a preliminary analysis of those proteins.Meanwhile, we also have cloned the two new molecular chaperone gene, which are linked to expression vector and have carried on the expression successfully.These experimental work will provide the possibility of seeking new corresponding effect protein later as well as their affection in the host cell, and lay the foundation of finally explanation of the pathogenesis mechanism of EHEC O157:H7.