重组质粒pgex.docVIP

重组质粒pGEX-6P-1/Sj-FABPc的构建及在大肠杆菌中的表达赵 巍1 苏 川2 吴海玮2 胡雪梅2 沈 蕾2?王荣芝2马 磊2 陈淑贞SUP.2 sup 张兆松2 【摘 要】 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pGEM-T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酶对融合蛋白进行解离，获得纯化的14kDa Sj-FABPc 。SDS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。【关键词】 日本血吸虫；脂肪酸结合蛋白；基因克隆；重组抗原；融合表达CONSTRUCTION OF RECOMBINANT pGEX-6P-1/Sj-FABPc AND EXPRESSION IN E.coli Zhao wei1 Su Chuan2 Wu Haiwei2 Hu Xuemei2 Shen Lei2 Zhang Zhaosong2 et al 1 Department of Biology ,Ningxia Medical College ,Yinchuan 750004 2 Institute of Medical Molecular Biology ,Nanjing Medical University, Nanjing 210029【Abstract】 Objective To obtain recombinant protein Sj-FABPc by gene engineering .Methods The gene fragment of Sj-FABPc was amplified by PCR from the adult 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 expressed and characterized. The fusion protein was purified by chromatography using Glutathion SepharoseTM 4B and was digested by the PreScissionTM 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 constructed and expressed as rSj-FABPc in E.coli BL21, the yield of which was around 10.52 mg/L. The rSj-FABPc can be recognized by the rabbit serum with Schistosoma japonicum infection using western blotting. Conclusions The recombinant fusion protein could be expressed efficiently and had good antigenicity.?【Key words】 Schistosoma japonicum, Fatty acid binding protein (FABP), gene cloning, Recombinant antigen, fusion expression??? 目前，血吸虫病防治的主要手段是采用吡喹酮进行化疗。但由于药物本身价格高，而且在一些反