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目的构建艰难梭菌(Clostridium difficile,C.difficile)毒素A羧基末端原核表达载体,优化诱导表达条件及纯化重组蛋白。方法利用聚合酶链式反应扩增C.difficile毒素A羧基末端基因序列,并将此序列转入pET-28b载体,构建pET-28b-tcdA重组表达载体,并将表达载体转化到BL21(DE3)感受态大肠埃希菌细胞中,分别在不同条件下进行诱导表达,获得最佳诱导表达条件后进行大量表达,最后用Ni柱对重组蛋白进行亲和纯化,得到纯化后的重组蛋白。结果成功构建了pET-28b-tcdA重组表达载体,其重组蛋白表达最佳诱导条件:菌液吸光度值取0.6、温度取25℃、IPTG终浓度取1.0mmol/L、诱导时间取10h。蛋白纯化咪唑磷酸盐洗脱液最佳浓度取200mmol/L。结论成功构建pET-28b-tcdA重组表达载体,在大肠埃希菌BL21(DE3)中可以有效表达,并获得高浓度重组蛋白,为进一步制备TcdA适配子奠定了一定实验室基础。
Objective To construct a prokaryotic expression vector for Clostridium difficile (C. difficile) toxin A, optimize expression conditions and purify recombinant protein. Methods The carboxy terminal sequence of C. difficile toxin A was amplified by polymerase chain reaction (PCR) and inserted into pET-28b vector to construct pET-28b-tcdA recombinant expression vector. The expression vector was transformed into BL21 (DE3) Competent Escherichia coli cells were induced under different conditions to obtain the best expression conditions for a large number of expression, and finally by recombinant Ni affinity purification of the recombinant protein to obtain the purified recombinant protein. Results The recombinant plasmid pET-28b-tcdA was successfully constructed. The optimal conditions for the recombinant protein expression were as follows: the absorbance of bacteria was 0.6, the temperature was 25 ℃, the final concentration of IPTG was 1.0mmol / L and the induction time was 10h. The optimum concentration of protein purification imidazole phosphate elution was 200mmol / L. Conclusion The recombinant plasmid pET-28b-tcdA was successfully constructed and expressed efficiently in Escherichia coli BL21 (DE3). High concentrations of the recombinant protein were obtained, which laid a good laboratory foundation for the further preparation of TcdA aptamers.