重组纤维连接蛋白EDA-EDB融合蛋白的分子克隆

Molecular cloning of recombinant fibronectin EDA and EDB fusion protein

目的构建重组pET28a-EDA-EDB质粒,制备重组纤维连接蛋白EDA-EDB融合蛋白。方法采用基因重组技术,将EDA、EDB基因片段连接,再将该重组基因插入pET28a表达载体,表达重组融合EDA-EDB蛋白后,利用6×His/Ni-NTA纯化系统进行纯化,免疫印迹法检测。结果成功连接EDA、EDB基因片段,重组pET28a-EDA-EDB质粒;在大肠杆菌BL21(DE3)中高度表达重组蛋白,获得较纯的重组蛋白,免疫印迹法证实为FN的组分。结论EDA-EDB重组蛋白能采用pET系统在大肠杆菌中表达,并可通过6×His/Ni-NTA纯化系统进行纯化而获得免疫原。

Objective Construct a recombinant plasmid pET28a-EDA-EDB,prepare the fusion EDA-EDB protein.Methods For the production of recombinant fibronectin EDA-EDB in Escherichia coli,the EDA and EDB segments were separated from pGEM2-EDA/EDB and recomposed with two additional amino acids,then cloned into the expression vector pET28a.pET system to express EDA-EDB fusion protein and6 × His/Ni-NTA system to purify it in a single step were used.Western blotting confirmed the purified protein.Results The EDA and EDB segments were ligated and inserted into pET28a vector.EDA-EDB fusion protein was highly expressed in Escherichia coli BL21(DE3).Afterwards,it was purified by Ni-NTA resin and verified by western blotting.Conclusion EDA-EDB fusion protein can be expressed in pET system and purified by6 × His/Ni-NTA system.