大鼠少突胶质细胞转录因子2基因真核表达载体的构建  

Construction of a rat oligodendrocyte transcription factor 2 eukaryotic expression vector

作　　者：欧阳长杰[1] 段线花[1] 戚举星[1] 曲德伟[1] 

机构地区：[1]徐州医学院人体解剖学教研室,江苏省徐州市221002

出　　处：《中国组织工程研究》2012年第42期7871-7876,共6页Chinese Journal of Tissue Engineering Research

基　　金：徐州市科技发展基金(XF10C079);徐州医学院院长专项人才科研基金(08KJZ04);徐州医学院科研课题(2010KJ17)~~

摘　　要：背景:碱性螺旋-环-螺旋转录因子少突胶质细胞转录因子2在脊髓的少突胶质细胞的分化过程中起着关键性的作用。目的:构建大鼠少突胶质细胞转录因子2基因重组真核表达载体,观察其在真核细胞内的表达。方法:从新生大鼠脊髓组织中提取总RNA,采用RT-PCR方法获得目的基因片段,克隆至pGEM-T载体中。经测序确证后将少突胶质细胞转录因子2cDNA片段与pEGFP-N1载体定向连接。将鉴定正确的重组体pEGFP-N1-Olig2以脂质体法转染至COS-7细胞中,反转录PCR鉴定少突胶质细胞转录因子2mRNA的表达,免疫印迹法检测少突胶质细胞转录因子2蛋白质表达水平。结果与结论:克隆了少突胶质细胞转录因子2的cDNA,并构建了其真核表达载体pEGFP-N1-Olig2,经限制性内切酶酶切鉴定及测序分析证实了其序列的正确性;转染72h时免疫印迹分析显示,COS-7/pEGFP-N1-Olig2实验组COS-7细胞表达Olig2-GFP融合蛋白。提示实验成功构建pEGFP-N1-Olig2真核表达载体,并且在COS-7细胞中得到高效表达。BACKGROUND：The basic helix-loop-helix transcription factor oligodendrocytes transcription factor 2 is a key regulator for the differentiation of oligodendrocyte lineage cells during development.OBJECTIVE：To construct the recombinant eukaryotic expression vector of rat oligodendrocyte transcription factor 2,and to investigate its expression in eukaryocytes.METHODS：Oligodendrocyte transcription factor 2 cDNA fragments were cloned by reverse transcription-PCR with the total RNA from neonatal rat spinal cord as the template,and subsequently cloned into pGEM-T vector.The oligodendrocyte transcription factor 2 fragment with its sequence confirmed was then cloned into pEGFP-N1 vector directionally.The right recombinant was transfected into COS-7 cells by lipofectamine 2000.The expression of oligodendrocyte transcription factor 2 in COS-7 cells was detected by reverse transcription-PCR and immunoblot analysis.RESULTS AND CONCLUSION：Oligodendrocyte transcription factor 2 cDNA was cloned,and the correct pEGFP-N1-Olig2 cloning was verified by restriction endonuclease digestion and sequencing.The Western blot analysis indicated that Olig2-GFP fusion protein was expressed in COS-7/pEGFP-N1-Olig2 cells at 72 hours after transfection.pEGFP-N1-Olig2 was constructed successfully.Olig2-GFP fusion protein could be abundantly expressed in COS-7/pEGFP-N1-Olig2 cells.

关 键 词：少突胶质细胞转录因子2 重组 质粒 基因表达 转染 COS-7细胞 碱性螺旋-环-螺旋转录因子 组织构建 组织工程 

分 类 号：R318[医药卫生—生物医学工程]