Cloning and expression of F18 fimbrial operon gene clusters from enterotoxigenic Escherichia coli and their bioactivity

Abstract:

The fed operon gene clusters with each size of 5.6kb，encoding the F18ab or F18ac fimbriae，was amplified respectively by high fidelity PCR using the genomic DNA templates from F18 fimbriae E. coli strains 107/86 or 2134P. The PCR products with the restriction enzyme sites at each end were digested and then cloned into the vector pET-22b(+)，the recombinant plamids with the inserts of both type of fed gene clusters were constructed and screened，further confirmed by the means of combination with restriction endonuclease analysis and sequencing. The both types of fimbriae F18ab and F18ac were expressed efficiently in the E. coli BL21(DE3) after proper concentration of IPTG induction. Expressed fimbriae were revealed and comfirmed by transmissible electromicroscope observation. The both fimbriae F18ab and F18ac were isolated and purified from the recombinant E. coli，and only a single major band of protein with size of approximately 15kDa was visualized in Coomassie blue-stained gels after SDS-PAGE. The rabbits sera with high titer of anti-F18 fimbriae were detected after being immunized with the purified F18ab or F18ac fimbriae. The results of combination of agglutination assay with Western blotting showed that the sera directed against both fimbriae F18ab and F18ac reacted positively with the F18 fimbriae from both wild E. coli 107/86 and 2134P.Small intestine epithelial cells with F18 fimbriae receptors，which were from post-weaning piglets with the genotypes of FUT1 gene both M307GG and M307AG，were prepared and tested for the adherence of E. coli expressing F18 fimbriae under the microscopic examination. Adhesion and adhesion inhibition test showed both of the recombinant E. coli expressing F18ab or F18ac fimbriae respectively could adhere to the jejunal epithelial cells in vitro as E. coli 107/86 and 2134p did. The both of anti-sera directed against fimbriae F18ab or F18ac respectively can efficiently inhibit the fimbriae-mediated post-weaning piglet jejunal epithelial cells adherence to both the recombinant E. coli (expressing F18ab or F18ac fimbriae) and wild type E. coli (107/86 and 2134P).