肠毒素性大肠杆菌CS3菌毛呈现载体的构建

Construction of a display vector based on the CS3 pili of enterotoxigenic Escherichia coli

目的 构建大肠杆菌CS3菌毛呈现载体 ,实现外源表位在细菌表面的呈现。方法 通过对CS3亚基蛋白二级结构、抗原表位、亲水性及柔韧性的预测分析 ,确定外源表位的插入位点 ,重叠延伸PCR方法进行定点突变 ,将口蹄疫病毒VP1插入到CS3中以验证表面呈现能力 ;用重组菌腹腔注射免疫小鼠以探讨其抗原性。结果 在大肠杆菌CS3的 136位氨基酸残基后突变插入BamHⅠ酶切点构建成呈现载体 ,全细胞ELISA、电镜和免疫电镜观察等均表明外源表位在大肠杆菌表面得到了表达 ,而且可诱发机体产生针对CS3和外源表位的双重免疫应答。结论 该大肠杆菌CS3菌毛呈现载体可以实现外源表位的表面呈现 ,可望成为研制基因工程活菌疫苗的表达载体。

Objective To construct the display vector based on the CS3 pili of enterotoxigenic Escherichia coli. Methods The secondary structure antigen epitopes, hydrophilicity and flexibility of CS3 subunit were predicted with the Goldkey software. Based on the prediction, the site for inserting heterologous epitopes was chosen. Mutation was done using the overlapping extention PCR. The gene fragment coding for the VP1 of foot mouth disease virus (FMDV) was synthesized and inserted into CS3. The surface expression of hybrid protein was examined using whole cell ELISA, electron microscopy and immuno electron microscopy. Mice were immunized by injecting the recombinant bacteria intraperitoneally to evaluate the immunogenicity of the hybrid proteins. Results The VP1 of FMDV was displayed on the surface of the recombinant cells. The fusion proteins were expressed as hybrid pili. Mice produced antibody response against CS3 and the VP1 of FMDV. Conclusion The CS3 pili can be a vector to express the foreign epitopes on the surface of the recombinant cells, and it may probably be an expression vector for the construction of the live gene engineering vaccine.