| Type 1 pili (fimbriae) are the most common adhesive appendages of E. coli, which are filamentous, proteinaceous appendages approximately 5-7 nm wide and 1-2μm long. They are principally composed of a single repeating polypeptide unit called pilin which is arrayed helically to form a hollow-cored fiber. Although these structures were thought for some time to be composed exclusively of pilin, it is now known that at least three other minor components (termed the adhesin ) is responsible for the ability of type 1-piliated bacteria to bind to a variety of eukaryotic cells. Binding can be inhibited by mannose, alpha-linked mannose oligosaccharides, and certain mannose analogs, indicating that the receptor on the eukaryotic cell contains mannose or a closely related compound.Type 1 fimbriae are encoded by the chromosomally located fim gene cluster. In addition to the genes encoding the structural proteins (fimA, fimF, fimG, and fimH), two genes, fimB and fimE, encode regulatory proteins directing the phase-dependent expression of the fimA gene, i.e. , controlling the periodical shifts between fimbriated and non-fimbriated states. Two other genes, fimC and fimD, have been found to be necessary for transport and assembly of the fimbriae.Because of the high homology between human and avian E. coli type 1 fim gene cluster, 9 primers were designed according to those published sequence in Genbank. The fim gene cluster was amplified respectively for several fragments by PCR using the DNA template of wild type fimbriated E. coli YR10(O18). The PCR products were cloned into pUC18 or pUCm-T. pBEA was made by inserting an EcoR I/Kpn I-cut PCR fragment BEA generated from YR10(O18) with primers P1 and P2 into pUC18 cut with the same enzymes. pUCm-IC and pUCm-D were made by T/A cloning of PCR fragments IC and D with primers P3, P4 and P5, P6. pBC was made by inserting a Kpn I/BamH I-cut fragment from pUCm-IC into pBEA cut with the same enzymes. pBD was made by inserting a BamH I/Sal I-cut fragment from pUCm-D into pBC cut with the same enzymes. Fragment DFGH was linked by SOE PCR with primers P5, P9 and PCR fragments D'and FGH generated from YR10(O18) with primers P5, P7 and P8, P9. pBH was made by inserting DFGH BamH I/Sal I-cut fragment into pBC cut with the same enzymes.The vectors pBC, pBD and pBH were transformed into HB101 (fim-) respectively, and grown over night at 37℃, 200r/m. Then the cells were identified by SDS-PAGE, agglutination and electron micrographs. Compared with HB101(pBC),HB101(pBD) had a 18kDa protein(FimA) band and HB101(pBH) had 18kDa and 30 kDa protein(FimH) bands; HB101(pBC) didn't react with antipilus sera, while HB101(pBD) had weak reaction and HB101(pBH) had strong reaction; The agglutination with eukaryotic cells and yeast showed that, HB101(pBC) and HB101(pBD) were agglutinate negtive, but HB101(pBH) had stronger ability to agglutinate the eukaryotic cell and yeast, and the binding could be inhibited by mannose and antipilus sera; The electron micrographs showed that HB101(pBC) couldn't express type 1 fimbriae, but HB101(pBD) and HB101(pBH) were fimbriated. The pilus of HB101(pBD) were much less than HB101(pBH), but were very longer. These indicated that subunit FimD were prerequisite in expression and FimF, FimG and FimH were nonessential but had great importance in pilus number, length, and adhesin.The results of the work have done a great contribution to construct type 1 fimbriae surface display system and product genomic vaccine. |
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