pcDNA3．1／myc-His-DJ-1^M26I真核表达载体构建及其在NIH3T3细胞中表达  

Construction of Expression Vector pcDNA3.1/myc-His-DJ-1^M26I and Expression in NIH3T3 Cells

作　　者：张梅英[1] 王惟[1] 徐影琪[1] 赵越[1] 杨葳[1] 于萌[1] 郭晓冲[1] 秦英[1] 郑志红[1,2] 

机构地区：[1]中国医科大学实验动物部辽宁省转基因动物研究重点实验室实验动物生物工程与转基因技术应用重点实验室,沈阳110001 [2]中国医科大学病理学与病理生理学研究室,沈阳110001

出　　处：《实验动物与比较医学》2012年第2期111-115,共5页Laboratory Animal and Comparative Medicine

基　　金：辽宁省科技计划项目（编号：2009408001-1）

摘　　要：目的构建pcDNA3．1／myc-his-DJ-1和pcDNA3．1／myc-his-DJ-1^M26I重组表达载体，为进一步研究DJ-1^M26I基因的功能及建立转基因动物模型奠定基础。方法采用突变试剂盒将第26位氨基酸进行突变，分别构建pcDNA3．1／myc-his—DJ-1和pcDNA3．1／myc-his-DJ-1^M26I重组表达载体，采用脂质体转染的方法分别将pcDNA3．1／myc-his-DJ-1和pcDNA3．1／myc—his-DJ-1^M26I质粒转染NIH3T3细胞并用G418压力筛选稳定克隆，对获得的2种转染细胞在DNA水平、转录水平和蛋白质水平鉴定。结果pcDNA3．1／myc-his-DJ-1和pcDNA3．1／myc-his—DJ-1^M26I质粒转染NIH3T3细胞经G418筛选后，经PCR检测分别获得1个和3个阳性细胞克隆，RT-PCR及western blot方法进行DJ-1-His基因表达检测，结果均证明外源插入基因的表达，MTT实验结果初步证明转染DJ-1^M26I基因的NIH3T3阳性细胞组增殖速率低于正常NIH3T3细胞组（P〈0．05）。结论成功构建pcDNA3．1／myc-his-DJ-1和pcDNA3．1／myc-his-DJ-1^M26I重组表达质载体。Objective To construct the expression vector pcDNA3.1/myc-his-DJ-1 and pcDNA3.1/ myc-His-DJ-1^M26I to lay a foundation of further study on function of DJ-1^M26I and establish transgenic animals. Methods The 26th amino acid of DJ- 1 was mutated by gene mutation kit, then pcDNA3.1/mychis-D J-1 and pcDNA3.1/myc-His-DJ-1 ^M26Iwere constructed. The two plasmids were transfected into NIH3T3 cells respectively using liposome method, then the cells were selected with G418 and detected the DJ- 1 gene expression at DNA, transcription and protein levels. Results 1 clone of pcDNA3.1/mychis-D J-1 and 3 clones of pcDNA3.1/myc-his-DJ-1^M26I were obtained after G418 selection by PCR analysis. The expression of DJ-1-His were detected by RT-PCR and Western blot in pcDNA3.1/myc-his-DJ-1 and pcDNA3.1/myc-his-DJ-1^M26I transfected cells. The profieration rate of D J-1^M26I transfected cells was lower than normal NIH3T3 cells （P〈0.05） by MTT essay. Conclusion The expression vectors pcDNA3. 1/myc-his-DJ-1 and pcDNA3.1/myc-His-DJ-1^M26I were constructed successfully.

关 键 词：DJ-1 NIH3T3细胞 帕金森氏病 

分 类 号：R742.5[医药卫生—神经病学与精神病学] R-332[医药卫生—临床医学]