Construction Of Recombinant Strains With Ail Gene And The Invasion And Adherence Analysis Of These Strains

Yersinia enterocolitica was widely distributed in the natural world, was one of the few intestinal pathogenic bacteria which could grow in refrigerated temperature. It also was an important foodborne pathogenic bacteria; and had already been listed as a conventional detection project of import and export food in many contries. Yersinia enterocolitica was obviously pathogenic to humans and animals. Infection of Yersinia enterocolitica could manifest a series of various syndromes, besides gastrointestinal symptoms, still could cause diseases in cardiovascular system, respiratory system, bone connective tissue, even lead to death by systemic disease and sEpticemia.The common feature of pathogenic bacteria was the breakthrough to the defense mechanism of host, which was closely related with the survival features (intracellular survival) of bacterium. Invasion and intracellular survival were thought to be important aspects of the virulence of many pathogenic bacteria. The ability to invade eucaryotic cells was a property common to these. Especially for enteropathogenic bacteria, it appeared more important. Yersinia enterocolitica was a facultative intracellular pathogen. The ail gene of pathogenic Y. enterocolitica played an important role in the ability of these strains to invade intestinal Epithelial cells, which was thought to be important aspects of the virulence of Yersinia enterocolitica. In our previous research, the ail gene was classified into three types:the highpathogenicity strain A2; the lowpathogenicity strain A1; the new type A3.Invasive mechanism of Y. enterocolitica was very complex. It was a result of the integrated effects of various factors, including identifided and unidentified. Once some certain factors made a change, the entirety phenotype was likely to take a change. So the different nucleotide sequence of Yersnia enterocolitica ail gene may promote a variable phenotype. Objective1 To construct the recombinant strains of the different nucleotide sequence ail gene.2 To determine whether the sequence polymorphism of ail gene can cause a function change of Ail protein; and the relationship with the virulence differences in the different style strains by the adhesion and invasion assays to the cultured cells.3 To explore whether the rEpresentative strain IE419 of the new type ail gene has a change in adhesion and invasion capability and bacterial virulence.Methods1 We could obtain the three different sequences ail gene by PCR amplification. The PCR amplified coding region of ail gene was cloned into pMD18-T vector. We introduced the different ail gene into the same nonpathogenic strain Y40 and E.coil JM109 and chosed the positive recombinant colonies by PCR and sequencing.2 The positive recombinant strains which were proved properly connected with the vector were then tested individually in the adhesion and invasion assays to HEp-2 and HT-29 cells. Through that, we could compute the efficiency of adhesion and invasion of each colony strains to chose the positive strains with the phenotype of adhesion and invasion, and to determine whether the polymorphisms of ail gene sequence could influence the ability of adhesion and invasion to Epithelial cells. Especially the changes of A3 ail gene.Results1 We successfully constructed the recombinant plasmids pMD18-T-ail through PCR and sequencing. By transformation, we gained the recombinant strains JM109/pMD 18-T-ail andY40/pMD18-T-ail.2 The results of RT-PCR showed that the different nucleotide sequence ail gene were successful transcripted in all recombinant strains.3 The assays of the invasion and adhesion appeared that the A2 ail gene could confer an invasive phenotype on E.coil JM109 steadily. However, the E.coil recombinant strains containing the lowpathogenicity A1 and the new type A3 ail gene had a instability in invasive ability. There was only one invasion-positive cloning strain in all recombinant strains. And there was no diversity in the two cell lines.4 After interacted with the cultured cells, the distribution characteristics of the E.coil recombinant strains were different from the pathogenic Yersinia enterocolitica.5 We found that the nonpathogenic recombinant strains were unable to adhere or invade to either of tissue culture cells. In addition, the nonspecific adhesion, the inherent characteristics of nonpathogenic Yersnia enterocolitica, had a certain degree drop.Conclusion1 We successfully constructed the recombinant strains JM109/pMD18-T-ail and Y40/pMD18-T-ail.2 Through the adhesion and invasion assays to HEp-2 and HT-29 cells, we obtained the E.coil recombinant strains with the phenotype of attachment and invasion of cultured Epithelial cells.3 Sequence polymorphism of ail gene could have an important influence on the pathogenic virulence of the different biological serotype pathogenic strains. The lack of ail gene maybe a factor of the nonappearance of pathogenic capability in nonpathogenic Yersinia enterocolitica.4 In this study, the strains with A3 sequence pattern ail gene had no difference with the strain with A1 ail gene in the ability of adhesion and invasiveness.