Knockout And Virulence Test Of The Gene Coding For Stx2e Of Shiga Toxin-producing Escherichia Coli

Porcine edema disease （ED） caused by Shiga-like toxigenic Escherichia coli arethe most common causes of mortality of recently weaned pigs.No effective methodfor control of the disease is available. The organism has two types of virulent factors,fimbrial adhesins and Shiga-like toxin. Fimbria, the primary virulent factor of theorganism, is responsible for its adhesion to the enterocye receptor of piglets. TheShiga-like toxin produced by E. coli is absorbed through the intestinal wall, whichresults in the damage of the target tissue. E. coli causing edema disease attaches to thesmall intestine by means of fimbria F18. Shiga-like toxin2e（Stx2e） is also producedby above organism. However, effective vaccines against ED is not available presently.In this research the F18-positive Shiga toxin-producing E.coli is isolated fromJiangsu area. Stx2e genes partial deletion strain were constructed by recombinantgene technique. A set of primers that homologous to stx2e genes andFRT-PGK-gb2-neo-FRT genes, were designed and synthetized according to thepublished sequences of stx2e A （accession No.756-1201bp） andFRT-PGK-gb2-neo-FRT genes.The products are homologous to regions adjacent tothe Stx2e gene to be inactivated and template plasmids carrying kanamycin resistancegenes that are flanked by FRT （FLP recognition target） sites. E.coil strains of Stx2ewhich would be modified, was transformed with the expression plasmid pRedET,incubated at30℃; the expression of genes mediating pRedET was induced by theaddition of L-arabinose. After induction, the cells were prepared for electroporationand the PCR product carrying the homology arms was electroporated. Mutants of theStx2e gene were replaced by homologous recombination as kanamycin-resistantcolonies after the introduction into bacteria carrying a Red expression plasmid Stx2e.After selection and PCR identification reaction of recombinants, the resistance genelocated in the Stx2e:kan mutant was eliminated in the second recombination, by usingthe FLP recombinase, which acts on the directly repeated FRT sites flanking thekanamycin resistance gene. The study revealed that the Stx2e gene are not single copy in STEC genome, and lay foundation for the construction of a non-toxic F18＋STECtoxin genes deletion mutants.Mice of the Kunming strain were randomly divided into15groups with5animals ineach group. Mice of different groups were given each an wild type S451521of STECand its two mutant strains in different doses separately, highly respectively half lethaldose of determination, clinical symptom observation. The LD₅₀of wild type S451521,mutant strainsⅠand mutant strainsⅡwere shown to be5.99×10⁷，1.50×10⁸and3.78×10⁸cfu/mL respectively, the tissue lesion to mice of mutant strain was muchlighter than that of wild strain，and the virulece to Vero cell is2/5of wild strain.