Cloning, Expression Of F18 Fimbrial Operon Gene And Preliminary Study Of Their Bioactivity

Postweaning diarrhea(PWD) and edema disease (ED) are infectious diseases which are responsible for major economic losses in pig-raising industry wordwide. F18~+ Escherichia coli strains are known to cause edema disease (ED) and postweaning diarrhea(PWD) in piglets. A prerequisite for ED-PWD is bacterial colonization on piglet intestinal epithelial mucosal surface, which is initiated by the adherence of bacterial to the host cell surface and usually mediated by F18 fimbrial adhesions. A promising strategy to prevent ED-PWD is by hindering bacterial invasion with antibodies directed against adhesins that interrupt their recognition of common receptor structures. Pure fimbriae can elicit protective immune responses. But the Escherichia coli strains expressing F18 fimbriae is effected by many other factors when they are cultured in vitro, moreover they produce fimbriae poorly. No commercial vaccines are thus far Mscilable against ED-PWD presently.In this work, the fed gene cluster of 5.6 kb, encoding the F18ab and F18ac fimbriae, was amplified respectively by high fidelity PCR using the DNA template of F18~+ E. coli 107/86 or 2134p . The PCR products were digested by restriction enzymes and then cloned into prokaryotic expression vector pET-22b(+). The both type of fimbriae F18ab and F18ac were expressed efficiently by IPTG induction in host cell BL21 (DE3)E.coli . Expressed products were revealed and comfirmed by combination of SDS-PAGE and tranmissible electromicroscope.Two New Zealand rabbits were immunized respectively using F18ab and F18ac fimbriae purified from recombinant expression products in vitro. High levels of anti-F18 antibodies were detected in rabbits sera. The result of Western blotting showed that the sera directed against both fimbriae F18ab and F18ac reacted positively with the fimbriae F18 of E.coli 107/86.The Escherichia coli F18 receptor locus(ECF18R) has genetically been mapped to the halothane linkage group on porcine chromosome 6. Molecular analysis of DNA polymorphisms associated with resistantce of porcine to E.coli associated diseases facilitateed diagnostic assay to select pigs for breeding. The resistant and susceptible porcine genotypes of enterotoxigenic Escherichia coli F18 receptor were detected by PCR-RFLP. Furthermore, adhesion and adhesion inhibition test showed both of the two recombined strains expressing F18ab and F18ac fimbriae respectively can adhere to porcine jejunal epithelial cells as 107/86 and 2134p did. The both anti-sera directed against fimbriae F18ab and F18ac respectively can efficiently inhibit the fimbriae-mediated porcine jejunal epithelial cells adherence with recombinant E.coli expressing F18 fimbriae and wild type E.coli 107/86and 2134p.