艰难梭菌毒素A原核表达及重组蛋白纯化

Prokaryotic expression and recombinant protein purification of Clostridium difficile toxin A

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 the prokaryotic expression vector of Clostridium difficile（C.difficile）toxin A carboxyl terminus,optimize the induction conditions and purify the recombinant protein.MethodsPolymerase chain reaction method was used to amplify the sequence of C.difficiletoxin A carboxyl terminus.The gene sequences of C.difficile toxin A carboxyl terminus was inserted into pET-28 bvector to construct the recombinant expression vector pET-28b-tcdA.The recombinant vector pET-28b-tcdA was transformed into BL21（DE3）competent E.coli to express the recombinant toxin A protein under different induction conditions.A large number of recombinant proteins were expressed by BL21 E.coli with pET-28 btcdA vector under the optimum expression conditions.The recombinant protein was purified by using nickel column with affinity chromatography.Results The recombinant expression vector pET-28b-tcdA was successfully constructed,the optimum expression conditions of which in BL21 E.coli were as follows：A600nm was 0.6,incubation temperature was 25℃,the final concentration of Isopropylβ-D-1-thiogalactopyranoside（IPTG）was 1.0mmol/L and IPTG induction incubation time was 10 hours.The optimum eluent concentration of imidazole phosphate buffer was 200mmol/L during recombinant protein purification.Conclusion The recombinant pET-28b-tcdA vector was effectively expressed in BL21（DE3）E.coli and high concentration of recombinant proteins were acquired,which laid a foundation for the research on the aptamer of C.difficile toxin A.