法医学杂志

法医学杂志 ›› 2002, Vol. 0 ›› Issue (3): 140-143.

• 论文 • 上一篇    下一篇

重组纤维连接蛋白EDA-EDB融合蛋白的分子克隆

薛爱民,王华,叶荣,沈忆文,赵子琴   

  1. 复旦大学上海医学院法医学系,复旦大学上海医学院法医学系,复旦大学上海医学院法医学系,复旦大学上海医学院法医学系,复旦大学上海医学院法医学系 上海200032 ,上海200032 ,上海200032 ,上海200032 ,上海200032
  • 发布日期:2002-06-25 出版日期:2002-06-28

Molecular cloning of recombinant fibronectin EDA and EDB fusion protein

XUE AI-MIN,WANG HUA,YE RONG,ET AL.(DEPARTMENT OF FORENSIC MEDICINE,SHANGHAI MEDICAL SCHOOL,FUDAN UNIVERSITY,SHANGHAI200032)   

  • Online:2002-06-25 Published:2002-06-28

摘要： 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。

关键词: 纤维连接蛋白, EDA, EDB, 重组蛋白

Abstract: 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.

Key words: fibronectin, EDA\EDB domain, recombinant proteins