大鼠膜联蛋白A_2真核表达载体的构建及表达  

Construction and expression of eukaryotic expression vector for rat annexin Ⅱ

作　　者：刘庆梅[1] 怀娟[1] 张杨[1] 姜虹[2] 岳寿伟[1] 

机构地区：[1]山东大学齐鲁医院康复科,济南250012 [2]山东大学齐鲁医院教育部和卫生部心血管重构和功能研究重点实验室

出　　处：《中国康复医学杂志》2010年第10期937-941,952,共6页Chinese Journal of Rehabilitation Medicine

基　　金：国家自然科学基金资助课题(30872732;81071597);山东省自然科学基金资助课题(Z2007C04)

摘　　要：目的:克隆大鼠背根神经节膜联蛋白A2(annexin Ⅱ)的基因,构建真核表达载体并检测其表达。方法:以RT-PCR法扩增大鼠背根神经节(DRG)细胞中膜联蛋白A2(annexin Ⅱ)的基因,构建pGMT-Anxa2克隆载体,以此为模板插入flag标签扩增Anxa2-flag序列,将其定向插入真核表达载体pcDNA3.1(+),获得表达载体pcDNA3.1(+)-Anxa2-flag。以PCR、双酶切及序列测定法鉴定该质粒。以脂质体法将表达载体转染到HEK293细胞中,Western印迹和细胞免疫荧光检测annexin Ⅱ蛋白表达及细胞内定位。结果:PCR扩增Anxa2-flag片段大小与预期相同。通过PCR、HindⅢ和NotⅠ双酶切及测序鉴定,证明构建出了含大鼠annexin Ⅱ基因的真核表达载体。Western印迹和细胞免疫荧光的检测表明大鼠annexin Ⅱ基因稳定表达,并可在胞膜和胞质中广泛表达。结论:成功构建pcDNA3.1(+)-Anxa2-flag真核表达载体,并稳定表达蛋白,为研究annexin Ⅱ调控DRG伤害性感受传导的分子机制奠定基础。Objective：To obtain annexinⅡ gene,construct it＇s eukaryotic expression vector and observe it＇s expression.Method：According to the Genebank,the coding sequence of rat annexinⅡ gene Anxa2 was cloned by RT-PCR method from rat dorsal root ganglion（DRG） cells,and pGMT-Anxa2 was constructed,according to which,Anxa2-flag was amplified by inserting the flag-tagged sequences to the C-terminal.Then Anxa2-flag was ligated into the eukaryotic expression vector pcDNA3.1（＋）.The vector was evaluated by HindⅢ and NotⅠdouble enzyme incision,PCR and sequence analysis.Recombinant plasmid pcDNA3.1（＋）-Anxa2 was transfected into HEK293 cells by using lipofectamine 2000,while Western blot and immunofluorescence were used to detect the expression and localization of rat annexinⅡ protein in the transfected cells in vitro.Result：The size of PCR amplified product of Anxa2-flag was consistent with the expected fragment.Recombinant plasmid pcDNA3.1（＋）-Anxa2-flag was proved to be correct by HindⅢ and NotⅠdouble enzyme incision,PCR and sequence analysis.Western blot and immunofluorescence results indicated that rat annexin Ⅱ protein expressed stably in HEK293 cell,and diffusely distributed throughout plasma membrane and cytoplasm.Conclusion：The eukaryotic expression vector pcDNA3.1（＋）-Anxa2-flag was constructed successfully,which could effectively expressed in HEK293.This will facilitate the further study of molecular mechanism of annexin Ⅱ in DRG＇s nociceptive pain conduction.

关 键 词：背根神经节 膜联蛋白A2 真核表达载体 WESTERN印迹 免疫荧光 

分 类 号：R346[医药卫生—基础医学]