›› 2010, Vol. 41 ›› Issue (1): 18-21.doi: 10.3969/j.issn.0529-1356.2010.01.004

• 论著 • 上一篇    下一篇

rAAV2-EM>hGAD/EM>65重组载体的构建和功能检测

郑德宇1,2;赵君朋2;赵焕英2;赵春礼2;段德义2;徐群渊2*   

  1. 1. 辽宁医学院解剖学教研室,锦州121001; 2. 首都医科大学神经科学研究所,北京100069
  • 收稿日期:2009-02-16 修回日期:2009-03-26 出版日期:2010-02-06
  • 通讯作者: 徐群渊2

Construction and function of the recombinant vector expressing human glutamic acid decarboxylase 65

  1. 1.Department of Anatamy, Liaoning Medical University, Liaoning Jinzhou121001, China;2.Beijing Institute for Neuroscience Capital Medical University, Beijing100069, China
  • Received:2009-02-16 Revised:2009-03-26 Online:2010-02-06
  • Contact: XU Qun-yuan

关键词: 谷氨酸脱羧酶2, 腺相关病毒载体, 基因克隆, 反转录\|聚合酶链式反应,

Abstract: Objective To construct the recombinant rAAV2-hGAD65 vector and detect its function both in vitro and in vivo. Methods The cDNA of human glutamic acid decarboxylase 2 (hGAD65) gene, which was one of gamma-aminobutyric acid (GABA) synthetase, cloned by the method of RT-PCR, was subcloned into the adeno-associated virus (AAV) vector and formed the recombinant vector of AAV-hGAD65 (rAAV2-hGAD65). The recombinant vector was packaged by the AAV Helper-Free System and its titer was detected. The primary fibroblast, cultured from the rat lung, was transfected by the rAAV2-hGAD65. The expression of the hGAD65 in fibroblast was detected by immunohistochemical method and the level of GABA in the media was assayed by high performance liquid chromatograph (HPLC). EM>In vivo/EM>, the hGAD65 was detected by immunohistochemical method in STN and the concentration of the GABA in the reticular part of substantia nigra (SNr) was assayed by HPLC after the rAAV2-hGAD65 was injected into the subthalamic nucleus (STN) by the stereotaxic method. Results The sequence of hGAD65 cDNA was in accordance with that in the Genebank. The amino acid sequence of hGAD65 had no mutation and the titer of rAAV2-hGAD65 was reached 4.5 ×10SUP>11 /SUP>per milliliter. The efficiency of infection to the rat primary firoblasts was 80%, and the concentration of GABA in the media of fibroblasts was (45.66±6.07)nmol/L per liter. EM>In vivo/EM>, hGAD65 could be detected in STN, and the concentrateion of the GABA in the SNr was increased from (5.66±1.07)nmol/g to (12.66±2.59)nmol/g.Conclusion The cDNA of hGAD65 was cloned by RT-PCR and the recombinant vector of rAAV2-hGAD65 was constructed. The AAV can infect the primary fibroblast in vitro and the hGAD65 can catalyse the glutamic acic to GABA. In vivo, the concentration of GABA in the SNr was heighten after the rAAV2-hGA

Key words: Glutamic acid decarboxylase 2(hGAD65), Adeno-associated virus vector, Gene clone, RT\|PCR, Human

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