大鼠SERCA2a启动子虫荧光素酶质粒的构建、鉴定和转染  

CONSTRUCTION,IDENTIFICATION AND TRANSFECTION OF PLASMID pGL3-SERCA2a PROMOTER

作　　者：张能锋[1] 祝宝华[2] 

机构地区：[1]东南大学临床医学院,江苏南京210009 [2]东南大学医学院附属扬州医院心内科,江苏扬州225001

出　　处：《实用临床医药杂志》2006年第2期40-43,56,共5页Journal of Clinical Medicine in Practice

基　　金：江苏省135工程医学重点人才基金资助项目(RC2002016)

摘　　要：目的构建大鼠SERCA2a启动子-虫荧光素酶质粒,并进行鉴定和转染检测。方法PCR法扩增出含SER-CA2a启动子基因的DNA大片段,经pGEM-T esay载体连接后酶切获得目的基因,亚克隆到pGL3-Basic载体中;酶切及测序鉴定;用Lipofectin Reagent转染到心肌细胞中,细胞培养后裂解并作荧光检测。结果构建出了含虫荧光素酶基因的质粒,酶切及测序鉴定插入子为大鼠SERCA2a启动子,转染细胞后检测到荧光。结论成功构建了大鼠SERCA2a启动子-虫荧光素酶质粒,为SERCA2a基因调控等研究提供了工具和基础。Objective To construct an eukaryotic expression plasmid pGL3-SERCA2a promoter which contains a reporter gene encoding luciferase for exploring the role of SERCA2a promoter in ischemic myocardium, Methods Because of the specific structure of sarcoplasmic reticulum Ca^2＋-ATPase （SERCA2a） promoter, it is difficult to directly get a appropriate DNA fragment through the method of PCR. So a long fragment of SERCA2a promoter containing purposed DNA fragment was amplified by PCR from SD rat heart, and it was ligated with pGEM-T esay vector for cutting the long fragment into ideal fragment and sequencing. Then we constructed the pGL3-SER- CA2a Promoter by inserting the appropriate DNA fragment into pGL3-Basic vector. After identifi- cation of the positive clone by digestion of restriction endonuclease, the plasmid was transfected into rat myocardial cell to test its activation. Results DNA sequencing and restriction endonuclease analysis demonstrated that the sequence of the recombinant plasmid pGL3-SERCA2a Promoter was correct. The transfection experiment confirmed the level of its activation was significantly higher than that of controls. Conclusion The plasmid pGL3-SERCA2a promoter is successfully constructed and can be used in studying the function of SERCA2a promoter.

关 键 词：SERCA2a启动子 报道基因 质粒 转染 

分 类 号：R446[医药卫生—诊断学]