摘要
目的:克隆先天性长QT综合征KCNE1基因,并构建真核表达载体。方法:从人胎盘组织中提取总RNA,经逆转录-聚合酶链式反应(RT-PCR)扩增出KCNE1基因;采用T-A克隆法,将KCNE1基因克隆入pCR2.1 TOPO载体中,经限制性酶切基因重组后,构建真核表达载体pEGFP-N1-KCNE1,经DNA测序鉴定,用E ffectene转染试剂介导转染HEK293细胞。结果:采用基因克隆和重组法,得到了KCNE1真核表达载体pEGFP-N1-KCNE1,并在HEK293细胞中得到了表达。结论:KCNE1基因的克隆及其真核表达载体的构建和表达为进一步的功能研究奠定了基础。
Objective： To construct the eurokaryotic expression vector of KCNE1 gene and express recombinant KCNE1 in HEK293 cells. Methods ： Human KCNE1 gene fragment was amplified from human placenta total RNA by RT-PCR and cloned into the vector of pCR2.1 TOPO by means of T-A cloning. KCNE1 cDNA was obtained from pCR2.1-KCNE1 by restriction enzyme digestion and inserted into the same restriction site of pEGFP-N1. Thus pEGFP-N1-KCNE1 was constructed and transfected into HEK293 cells with Effectene transfection reagent. Results：The eukaryotic expression vector pEGFP- N1-KCNE1 was successfully constructed by gene cloning and recombinant method and expressed in HEK293 cells. Conclusion ：The cloning of KCNE1 gene and the construction and expression of its eukaryotic expression vector may shed some light on further functional study of KCNE1 gene.
出处
《医学研究生学报》
CAS
2007年第11期1126-1129,I0003,共5页
Journal of Medical Postgraduates
基金
国家自然科学基金资助项目(批准号:30170377)
国家科技攻关计划863项目资助(批准号:2002BA711A07)
