You Position: Home > Paper
Amplification of human GAD67 gene and construction of its eukaryotic expression vector pEGFPC2-GAD67
( views:275, downloads:12 )
- Author:
- No author available
- Journal Title:
- CHINESE JOURNAL OF NEUROMEDICINE
- Issue:
- 11
- DOI:
- 10.3760/cma.j.issn.1671-8925.2006.11.004
- Key Word:
- 谷氨酸脱羧酶(GAD)67;克隆;真核表达质粒
Abstract： 目的 扩增人类谷氨酸脱羧酶(GAD)67基因,并构建pEGFP-C2-GAD67真核表达载体.方法 应用基因重组技术,采用反转录PCR方法对来自人类脑组织cDNA中编码GAD67的基因片段进行扩增,插入PUC57克隆载体,经Xho Ⅰ和BamH Ⅰ双酶切出GAD67基因片段,再克隆入真核表达载体pEGFP-C2后构建pEGFP-C2-GAD67重组载体,经PCR扩增、酶切后测序鉴定. 结果 人类GAD67基因扩增产物大小为1 785 bp,编码594个氨基酸残基.重组真核表达载体经酶切鉴定表明为正确重组子,PCR正确扩增出目的条带.与GenBank中人类GAD67基因序列比较,核苷酸和氨基酸水平的同源性分别达99.78%和99.66%. 结论 人类GAD67基因克隆后,可成功构建真核表达载体pEGFP-C2-GAD67,为进一步该基因转染神经干细胞移植治疗癫痫提供了实验基础.
- [1]刘智良,徐如祥,姚志彬,等.GABAB受体对大鼠海马CA1区锥体细胞突触传递的作用[J].2006,(9).
- [2]Loetscher E,Kalkman HO.GAD(67): the link between the GABA-deficit hypothesis and the dopaminergic- and glutamatergic theories of psychosis.[J].2003,110(7).
- [3]Istvan Mody.The GAD-given Right of Dentate Gyrus Granule Cells to Become GABAergic[J].2002,2(5).
- [4]M G, Erlander,N J, Tillakaratne,S, Feldblum,etc.Two genes encode distinct glutamate decarboxylases.[J].1991,7(1).
- [5]F·奥斯伯.精编分子生物学实验指南[M].2000.
- [6]Allen NJ,Karadottir R,Attwell D.Reversal or reduction of glutamate and GABA transport in CNS pathology and therapy.[J].2004,449(2).
- [7]Andrade-da-Costa BL,Hokoc JN,de-Mello FG.Transporter-mediated GABA release induced by excitatory amino acid agonist is associated with GAD-67 but not GAD-65 immunoreactive cells of the primate retina.[J].2000,863(1).
- [8]姜泊主编,张亚历主编,周殿元主编.分子生物学常用实验方法[M].1997.