CT-1及TTC的GST融合蛋白表达质粒的构建及表达

Construction of GST fusion protein expression plasmid of the cardiotrophin-1 and tetanus toxin C fragment and its expression

目的构建心肌营养素-1(CT-1)和破伤风外毒素C片段(TTC)重组融合蛋白表达质粒。方法利用PCR、TA克隆等技术将CT-1及TTC基因克隆并连接到pGEX-4T-3,再做酶切鉴定、序列测定。在不同温度不同时间,通过IPTG诱导pGEX-CT-1/TTC在DH5α大肠杆菌中表达,SDS-PAGE分析表达量和可溶性蛋白GST-CT-1/TTC比例。结果构建的pGEX-CT-1/TTC酶切结果可见606 bp1、356 bp大小的片段,与预期结果一致,测序结果表明序列正确。通过IPTG诱导,发现35℃诱导4 h,目的蛋白GST-CT-1/TTC表达量占全菌的1/5,可溶性蛋白与包涵体比为2/5。结论本实验成功构建了pGEX-CT-1/TTC重组融合质粒,为下一步将其用于脊髓损伤的治疗研究打下了基础。

Objective To construct the GST fusion protein expression plasmid of cardiotrophin-1 （CT-1） and tetanus toxin C fragment （TTC）. Methods CT-1 and TTC genes were cloned and ligated to pGEX-4T-3 plasmid by PCR and ToA cloning. The recombinant plasmid was digested to insure the inserted gene, and then sent to the professional company for sequencing. Since the pGEX-CT-1/TTC expression was induced by IPTG in E. coli at different temperature and various times, the expression level and the proportion of the soluble protein were analyzed by SDS-PAGE. Results After digestion of the recombinant plasmid pGEX-4T3, two gene fragments of 606 bp and 1 356 bp were found, which was concord with the that of expection. After the induction of pGEX-CT-1/TTC-DH5α cells by IPTG at 35 ℃ for 4 h, the highest expression level of the recombinant protein was about 1/5 of the total cell proteins, and the soluble protein was about 2/5 of the fusion protein. Conclusion The recombinant fusion plasmid of pGEX-CT-1/TTC is constructed successfully, which provides foundation for therapy of the spine cord injury by cardiotrophin-1.