长QT综合征2型HERG基因绿色荧光真核表达载体的构建及表达

Construction of eukaryotic expression vector of congenital long QT syndrome related HERG gene and its expression in HEK293 cells

目的构建先天性长QT综合征2型HERG基因绿色荧光真核表达载体p EGFP-C2-HERG,并检测其于活体细胞中表达及定位。方法采用限制性内切酶双酶切法和基因重组技术将HERG基因片段构建到真核表达载体p EGFP-C2中,通过琼脂糖凝胶电泳和DNA测序验证;构建成功的p EGFP-C2-HERG真核表达载体用脂质体转染法转染HEK293细胞后通过蛋白免疫印迹法再次鉴定重组蛋白的表达,并通过激光共聚焦显微镜观察其荧光蛋白表达及定位。结果琼脂糖凝胶电泳证实新构建的真核表达载体p EGFP-C2-HERG碱基大小正确,DNA测序提示HERG基因碱基序列与模板一致,新构建表达载体成功转染入HEK293细胞并于细胞膜上成功表达。结论成功构建p EGFP-C2-HERG真核表达载体,转染入HEK293细胞并于细胞膜上成功表达通道蛋白;此方法构建的表达绿色荧光的真核表达载体为药物筛查、突变型基因的研究奠定了基础。

Objective To construct the type 2 long QT syndrome（LQT2） related human-ether-a-go-go-related gene（HERG） pEGFP- C2-HERG eukaryotic expression vectors and detect its expression and localization in alive cells. Methods The HERG sequence was subcloned into the pEGFP-C2 vectors using restriction enzymes and gene recombination technology. The recombinant plasmids pEGFP- C2-HERG were identified by agarose gel electrophoresis and sequencing. The pEGFP-C2-HERG was transfected into HEK293 cells with lipofectamine to observe the fluorescence expression and location. Results Agarose gel electrophoresis confirmed that the base size of new recombinant eukaryotic expression vector was correct, and that genetic base sequence was in accordance with the template u- sing DNA sequence. The pEGFP-C2-HERG was successfully transfected into HEK293 cells and expressed in the cell membrane. Con- clusion The pEGFP-C2-HERG green fluorescent protein eukaryotic expression vector could be successfully constructed and expressed in HEK293 cells, meanwhile HERG channel protein is expressed in the cell membrane, and the protocol of recombinant green fluores- cent eukaryotic expression vectors lay the foundation for drug screening and research of mutant gene.