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

Objective To construct the GST fusion protein expression plasmid of cardiotrophin-1(CT-1) and tetanus toxin C frag-(ment(TTC).) Methods CT-1 and TTC genes were cloned and ligated to pGEX-4T-3 plasmid by PCR and T-A 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.