| Enteroioxigenic Escherichia coli (ETEC) is a major etiological cause of diarrhea and death in postweaning pigs. These pathogens have two types of virulence factors, fimbrial adhesins and enterotoxins. The fimbriae enable the ETEC to colonize the small intestine of piglets by mediating adhesion to the microvilli of epithelial cells. The first fimbrial adhesin described for an ETEC strain isolated from an young pig with diarrhea was K88. Most ETEC strains from piglets with diarrhea possess more one of the fimbrial adhesins for K88、 K99、987P or F41; of which K88is the predominant fimbrial adhesin. Recent research suggested that, in addition to proven roles in secretory diarrhea, the heat-labile enterotoxin LT may also provide a competitive advantage for adherence to intestinal epithelial cells. Therefore, in this study, we focus on the following two parts: first one, cloning and expression of K88ab/K88ad fimbrial operon gene clusters and their bioactivity. The second, cloning and expression of LT gene and its contribution to adherence to intestinal epithelial cells. It is to build the foundation to determine the full complement of virulence factors for the intestinal colonization.Study one:the fae operon gene clusters with size of7.9Kb, encoding the K88fimbriae, was amplified respectively by high fidelity long-PCR using the genomic DNA templates of K88ab and K88ad fimbriae.E. coli strains. The PCR products with the restriction enzyme sites at each end were digested and then cloned into the vector pBR322, the recombinant plamids with the inserts of fae gene clusters were constructed and screened, further confirmed by the means of combination with restriction endonuclease analysis. Expressed fimbriae were revealed and comfirmed by transmissible electromicroscope observation. The fimbriae K88ab and K88ad were isolated and purified from the recombinant E.coli, and only a single major band of protein with size of approximately26KD was visualized in Coomassie blue-stained gels after SDS-PAGE. The results of combination of agglutination assay with Western blotting showed that the monoclonal antibody(Mab) directed against K88fimbriae reacted positively with the K88ab and K88ad fimbriae from the recombinant E. coli. Small intestine epithelial cell line IPEC-J2with K88fimbriae receptors were prepared and tested for the adherence and adhesion inhibition of E. coli expressing K88fimbriae. The results showed the recombinant E.coli expressing K88fimbriae could adhere to IPEC-J2in vitro as wild type E.coli did. The anti-sera directed against fimbriae K88can efficiently inhibit IPEC-J2adherence to the recombinant E. coli(expressing K88fimbriae)and wild type E. coli.Study two:The LT gene with size of1.1Kb, was amplified by PCR using the genomic DNA template of K88ac E. coli strain C83902. The PCR products with the restriction enzyme sites at each end were digested and then cloned into the vector pACYC184, the recombinant plamids with the inserts of LT gene were constructed and screened, further confirmed by the means of combination with restriction endonuclease analysis. Two well-characterized variants of LT, each bearing mutations of amino acid residue in the A subunit (A72R and R192G), were also constructed. The expression of LT protein was comfirmed by GM1-ELISA. Small intestine epithelial cell line IPEC-J2, which produce glycoprotein receptors for bacterial adhesions in vitro, were prepared and tested for the adherence of E. coli expressing LT or not. The experimental data demonstrated that elaboration of LT promotes a significant increase in E. coli adherence. Two well-characterized variants of LT were constrcted to prove that ADP-ribosylation activity is necessary to effect changes in bacterial adherence. Rp-cAMP, an inhibitor of protein kinase A; DDA, the adenylate cyclase antagonist and GM1, the receptor of LT were sufficient to abrogate LT’s ability to promote subsequent bacterial adherence. |
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