F18大肠杆菌黏附素受体结合域鉴定新方法

A novel identification method of the receptor-binding domain for FedF from pathogenic F18 fimbrial Escherichia coli

运用DNA重组技术将F18ab和F18ac大肠杆菌黏附素fedF亚单位内的第60～109位氨基酸残基区段(fedF1)和fedF全基因克隆入V型分泌系统MisL的载客结构域上,经DNA测序确证其正确阅读框后,含重组菌质粒pnirBMisL-fedF或pnirBMisL-fedF1经厌氧诱导后,与断奶仔猪小肠上皮细胞进行体外黏附试验。结果表明:FUT1基因M307位点中GG型和AG型仔猪小肠上皮细胞均能黏附上述重组菌,而AA型个体小肠上皮细胞则不能黏附。上述重组菌均能与兔抗F18ab菌毛FedF亚单位单因子高免血清发生玻板凝集反应。而FedF突变体FedF(M)的重组质粒菌则失去上述凝集和黏附特性。证明F18大肠杆菌黏附素fedF基因及其基因片段fedF1在大肠杆菌表面得到了功能性表达,F18ab和F18ac黏附素FedF直接介导F18大肠杆菌与易感仔猪小肠上皮细胞表面大分子受体黏附结合,fedF1为黏附素FedF的受体主要结合域,其His88、His99是FedF黏附素受体结合域的重要氨基酸残基。

Using the DNA manipulation,the diferent recombinants of pnirB MisL from the type V secretion system MisL with DNA inserts of fedF1（between amino acid residues 60 to 109 of fedF）,fedF（M）（both the positions 88th and 89th amino acid residues changed from Histidine to Alamine） or fedF from both F18ab and F18ac fimbrial E.coli were constructed,respectively.After inducing of IPTG at the anaerobic condition,the above recombinant E.coli DH5α with the different recombinants pnirBMisL-fedF1 and pnirBMisL-fedF were tested for their adhesion capability in vitro with the small intestinal epithelial cells from the post-weaned piglets.The results showed that all the recombinant bacteria with pnirBMisL-fedF1 or pnirBMisL-fedF could adhere well to the small intestinal epithelial cells with the FUT1 genotypes of both M307^GG and M307^AG,except genotype M307^AA.They all could agglutinate with the anti-rabbit sera against FedF subunit.But the recombinant bacteria with the pnirBMisL-fedF（M） mutants completely abolished the capability for both the antibody agglutination and receptor binding.The results confirmed that the receptor binding domain of FedF1,even FedF could be both secreted and surface displayed efficiently in a functional form in E.coli DH5α by using type V secretion system and that the recombinant E.coli carrying different recombinants pnirBMisL-fedF1 and pnirBMisL-fedF adhered to porcine intestinal epithelial cells via the FedF protein or FedF1 which is the major receptor binding region.His-88 and His-89 amino acid residues located in the FedF adhesin were important for the formation of the binding domain of the adhesin FedF of F18ab fimbriae.