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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 E. coli CS3 pilus as a carrier, to achieve the appearance of foreign epitopes on the bacterial surface. Methods The secondary epitopes, hydrophilicity and flexibility of CS3 subunit protein were predicted and analyzed. The insertion sites of exogenous epitopes were identified and the site-directed mutagenesis was performed by overlap extension PCR. VP1 of foot-and-mouth disease virus was inserted into CS3 In order to verify the surface rendering ability; recombinant mice intraperitoneal injection of immunized mice to explore its antigenicity. Results After the amino acid residue of Escherichia coli CS3 was mutated and inserted into BamH Ⅰ site, the recombinant vector was constructed. Whole cell ELISA, electron microscopy and immunoelectron microscopy showed that the foreign epitopes were expressed on the surface of E. coli and induced The body produces a dual immune response to CS3 and foreign epitopes. Conclusion The E. coli CS3 pili carrier can be used as the expression vector for the genetically engineered live vaccine.