中国组织工程研究

中国组织工程研究 ›› 2012, Vol. 16 ›› Issue (42): 7871-7876.doi: 10.3969/j.issn.2095-4344.2012.42.016

• 组织构建实验造模 experimental modeling in tissue construction • 上一篇    下一篇

大鼠少突胶质细胞转录因子2基因真核表达载体的构建

欧阳长杰，段线花，戚举星，曲德伟   

  1. 徐州医学院人体解剖学教研室，江苏省徐州市 221002
  • 收稿日期:2012-07-16 修回日期:2012-07-19 出版日期:2012-10-14 发布日期:2012-10-14
  • 通讯作者: 曲德伟，博士，副教授，徐州医学院人体解剖学教研室，江苏省徐州市 221002
  • 作者简介:欧阳长杰★，男，1976年生，江苏省徐州市人，汉族，2008年徐州医学院毕业，硕士，讲师，主要从事神经干细胞的基因治疗的基础研究工作。 Ouyang7605@163.com

Construction of a rat oligodendrocyte transcription factor 2 eukaryotic expression vector

Ouyang Chang-jie, Duan Xian-hua, Qi Ju-xing, Qu De-wei   

  1. Department of Human Anatomy, Xuzhou Medical College, Xuzhou 221002, Jiangsu Province, China
  • Received:2012-07-16 Revised:2012-07-19 Online:2012-10-14 Published:2012-10-14
  • Contact: Qu De-wei, Doctor, Associate professor, Department of Human Anatomy, Xuzhou Medical College, Xuzhou 221002, Jiangsu Province, China
  • About author:Ouyang Chang-jie★, Master, Lecturer, Department of Human Anatomy, Xuzhou Medical College, Xuzhou 221002, Jiangsu Province, China Ouyang7605@163.com

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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2 cDNA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2 mRNA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72 h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。

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

Abstract:

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.

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