| F18 fimbrial Escherichia coli is the main pathogen causing porcine postweaning diarrhea (PWD) and pig edema disease(ED). The bacteria have two serotypes of F18ab and F18ac. The prerequisite for PWD and ED is colonization on the host intestinal epithelial mucosal surface by the bacteria, which is mediated by the adhesins of F18 fimbriae. Some studies have showed that the FedF subunit is the adhesin of F18 fimbriae and that the receptor binding region of FedF is between amino acid residues 60 and 109. Using the type V secretion system MisL, the recombinant plasmids surface-displaying FedF adhesin and the receptor binding domain FedF1 in the E.coli DH5αwere constructed successfully. After anaerobic induction, the recombinant bacteria with pnirBMisL-fedF or pnirBMisL-fedF1 could agglutinate with the anti- rabbit sera against FedF subunit and adhere to the small intestinal epithelial cells well from the sensitive piglets. But the recombinant bacteria with the pnirBMisL-fedF(M) (both the 88th and 89th amino acid residues substituded from histidine to alanine in fedF subunit) completely abolished the agglutination characteristic and receptor adhesiveness. These results confirmed that the binding domain FedF1 and the adhesin FedF could be transported and displayed functionally on the surface of E.coli DH5αand the His-88 and His-89 amino acid residues which located in the FedF adhesin were critical for the formation of the binding domain of the adhesin FedF of F18 fimbriae.For further study the expression and application of F18 fimbrial adhesin in chromosome-plasmid balanced-lethal system, E.coli DH5α△asd deletion mutant was constructed by using Red recombination system. First, the chloramphenicol resistance (cat) gene flanked by homology extensions of asd gene was amplified by PCR. The PCR products were electro-transformed into E.coli DH5αstrain, with the help of Red recombinant system, the most part of asd gene was in vivo replaced by homology extensions connected with cat gene. E.coli DH5α(△asd::cat) deletion mutant with cat gene was selected by LB plate with DAP and chloramphenicol. The cat gene was then eliminated by using a helper plasmid, pCP20, encoding the FLp recombinase; the result recombinant bacterium was named E.coli DH5α△asd which lost the capability of growth on LB plate. The deletion mutant was recovered the capability of growth on LB plate when added with DAP. In addition, the function of the asd deletion mutant could be compensated by the plasmid expressing asd gene.Based on the flanking sequences of E.coli K-12 strain asd gene, a pair of primer designed were used to amplify the DH5α-derived asd gene by PCR. The asd gene was cloned into the above plasmid pnirBMisL-fedF and the plasmid pnirBMisl-fedF-asd was constructed with the inactivation of ampicillin resistance gene and the expression of asd gene at the same time. After the plasmid pnirBMisl-fedF-asd was transformed into E.coli DH5α△asd deletion mutant, the chromosome-plasmid balanced-lethal system was set up successfully based on the asd gene. This system is stable in the LB media for 20 generations tested without curing of the recombinant plasmid. The functionally surface display of F18 E.coli adhesin was proved by indirect fluorescent antibody technique and adhesion assay of porcine small intestinal epithelial cell.The construction of the asd gene labeled chromosome-plasmid balanced lethal system surface-displaying the FedF adhesin without antibiotic resistance gene provides useful tool to study the subunit vaccine to prevent PWD and ED.
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