重组质粒pGEX-6P-1/Sj-FABPc的构建及在大肠杆菌中的表达

目的构建基因工程菌株,获得重组蛋白Sj-FABPc(日本血吸虫脂肪酸结合蛋白).方法用PCR法从日本血吸虫cDNA文库中扩增Sj-FABPc基因片段,再将该片段重组于pGEMT中并进行DNA测序鉴定,经酶切后将目的片段构建成重组质粒pGEX-6P-1/Sj-FABPc,转化于大肠杆菌BL21,IPTG诱导表达.用Glutathione SepharoseTM 4B亲和层析柱对表达产物进行纯化,PreScissionTM Protease酶对融合蛋白进行分离,获得纯化的14kDa Sj-FABPc.SDSPAGE和Western blot方法对表达产物进行鉴定.结果获得pGEX-6P-1/Sj-FABPc菌株,分离纯化出14kDa Sj-FABPc,表达量为10.52mg/L.Western blot显示,表达蛋白能被日本血吸虫免疫兔血清识别.结论重组蛋白Sj-FABPc可高效表达并有良好的抗原性.

CONSTRUCTION OF RECOMBINANT pGEX-6P-1/Sj-FABPc AND EXPRESSION IN E. coli

Objective To obtain recombinant protein Sj-FABPc by gene engineering. Methods The gene fragment of Sj-FABPc was amplified by PCR from the worm cDNA library of Schistosoma japonicum and then subcloned into pGEM-T vector. The gene sequence was identified and the target fragment was restrictedly digested and subcloned into expression vector pGEX-6p-1. The protein is exprssed and characterized. The fusion protein was purified by chromatography using Glutathion Sepharose~(TM) 4B and was digested by the PreScission~(TM) Protease. The 14kDa Sj-FABPc can be obtained. The antigenicity of rSj-FABPc has been demonstrated by Western Blot. Results The pGEX-6P-1/Sj-FABPc was obtained and can express rSj-FABPc the yield of which was around 10.52 ml/L. The rSj-FABPc, can be recognized by the rabbit serum with schistosoma japonicum immunity using western blotting. Conclusion The recombinant fusion protein could be expressed efficiently and had good antigenicity.