Ⅴ型分泌系统菌体表面展示F18大肠杆菌黏附素及其受体结合位点的确定

Display of the F18 fimbrial adhesin on the bacterial surface using type Ⅴ secretion system and characterization of the binding domain of the adhesin

摘要:

自主转运蛋白(V型分泌系统)的β结构域已被证明可以将异源性多肽展示在细菌表面。运用DNA重组技术优化构建V型分泌系统MisL并在菌体表面展示F18大肠杆菌黏附素FedF及其受体结合域FedF1。含重组质粒pnirBMisL-fedF或pnirBMisL-fedF1大肠杆菌(E.coli)DH5α经厌氧诱导后,分别与兔抗F18ab菌毛FedF亚单位单因子血清和F18大肠杆菌黏附素受体易感性仔猪的小肠上皮细胞做玻板凝集试验和体外黏附试验,结果表明上述两株诱导表达重组菌与FedF抗血清发生明显的凝集反应,且能较好地黏附于F18大肠杆菌黏附素受体易感性仔猪小肠上皮细胞。而菌体表面展示F18大肠杆菌黏附素FedF突变体FedF(M)(黏附素受体结合域第88和89位组氨酸残基双突变为丙氨酸)的重组菌则失去上述凝集和黏附特性。以上试验结果说明,F18大肠杆菌黏附素FedF及其受体结合域FedF1在大肠杆菌表面得到了功能性表达,并进一步证明了位于FedF受体结合域内第88和89位组氨酸残基对FedF受体结合域的形成至关重要。

Abstract:

The β domains of autotransporters (also called type Ⅴ secretion system) have been demonstrated to be feasible tools to display foreign passenger domain on the bacterial surface. In the present work, using the DNA manipulation the type Ⅴ secretion system MisL displaying the binding doamin FedF1 of the F18ab fimbrial adhesion was constructed, and the full length adhesin FedF of F18ab fimbriae and the FedF mutant (with the 88th and 89th amino acid residues changed from Histadine to Alamine) on the surface of E. coli DH5α, respectively. The recombinant E. coli DH5α with the different recombinant plasmids of pnirBMisL-fedF1, pnirBMisL-fedF and pnirBMisL-fedF(M) were induced at anaerobic conditions. After induction, the binding doamin FedF1 of the FedF and adhesin FedF displayed on the surface of E. coli DH5α were tested for their agglutination and adhesion capability with the anti-rabbit sera against FedF subunit or the small intestinal epithelial cells from susceptible piglets. The results showed that the pnirBMisL-fedF1 or pnirBMisL-fedF recombinant bacteria could agglutinate with the anti-rabbit sera against FedF subunit and adhere to the small intestinal epithelial cells well. But the recombinant bacterial strain with the recombinant plasmid pnirBMisL-fedF(M) completely abolished the agglutination characteristic and receptor adhesiveness. These results confirmed that the binding doamin FedF1 of the F18ab fimbrial adhesin and the adhesin FedF of F18ab fimbriae could be transported and displayed functionally on the surface of E. coli DH5α by using type Ⅴ secretion system and the 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.