【摘要】 目的获得Asp-Asp-Asp-Asp-Lys序列+牛EKL的cDNA序列,实现EKL基因在大肠杆菌中的融合表达和自切割。方法用Trizol法从牛十二指肠组织中提取总RNA,利用RT-PCR技术扩增其cDNA片段,将此片段克隆于表达载体pGEX-2T,并在其前引入肠激酶(EK)识别序列。结果所表达蛋白经SDS-PAGE分析,相对分子质量约为61 000,经GST-Sepharose亲和色谱柱纯化后得到单一蛋白,SDS-PAGE显示单一条带,用微量EK引发自切割,切断GST标签蛋白和Asp-Asp-Asp-Asp-Lys序列,采用对氨基苯甲脒-Sepharose亲和色谱获得了轻链EK的单体。结论成功地克隆、表达了牛EK轻链基因,采用自激活、切割获得了EK单体,每1 L培养基获得EK5 mg,为进一步进行重组牛EK活性的研究及应用奠定了基础。更多还原

【Abstract】 Purpose The sequence of bovine EKL gene with Asp-Asp-Asp-Asp-Lys sequence in the N-terminus was obtained and analyzed,and the fusion protein of the EKL-GST was produced in E.coli.Methods The fragment of bovine enterokinase light chain cDNA was obtained by RT-PCR from bovine′s duodenal mucosa,then cloned into the pMD18-T cloning vector and sequenced.Compared with the sequence deposited in GenBank,the cloned gene sequence is correct.Then the interested gene fragment was inserted into the pGEX-2T expression plasmid.The recombinant vector pGEX-rEKL was transformed into E.coli BL21 and induced by IPTG.Results SDS-PAGE analysis indicated that the target product was about 61 kDa.The research successfully clones and expresses EKL-GST fusion protein in E.coli.Selt-activation by microamount cutting the fusion protein,and purified the EKL by minobenzene-Sepharose.Conclusion This investigation would be able to lay a foundation for enterokinase activity research and further application of expression products on a large scale.更多还原