摘要
Objective Glutamic acid decarboxylase 2（GAD65） is a gamma-aminobutyric acid（GABA） synthetase.This study aimed to construct a recombinant lentivirus-rGAD65（rLV-rGAD65） vector containing the cDNA of rat GAD65（rGAD65） and assess its functional activity in vitro and in vivo.Methods cDNA of rGAD65 was amplified by RT-PCR and subcloned into the LV vector,forming the rLV-GFP-rGAD65 plasmid.The recombinant lentivirus particles（rLVrGAD65） were packaged by the LV Helper-Free System and the titer was measured.Primary rat lung fibroblasts were transfected with rLV-rGAD65.The expression of rGAD65 in fibroblasts was detected by immunocytochemistry and western blot and the level of GABA in the medium was assessed by high-performance liquid chromatograph（HPLC）.In vivo,rLV-rGAD65 was injected into the subthalamic nucleus（STN） of Sprague-Dawley rats using stereotaxic methods,and rGAD65 protein levels in the STN were assessed by immunohistochemistry and Western blot,while the GABA concentration in the substantia nigra pars reticulata（SNr） was assayed by HPLC.Results The sequence of rGAD65 cDNA was in accord with that in GenBank.The amino-acid sequence of rGAD65 had no mutations and the titer of rLVrGAD65 reached 6.8 × 108/mL.The efficiency of infection of fibroblasts was 80%,and the concentration of GABA in the medium was（48.14 ± 9.35） nmol/L.In vivo,rGAD65 expression was detected in the STN,and the concentration of GABA in the SNr increased from（5.95 ± 1.09） to（12.44 ± 3.79） nmol/g tissue.Conclusion The recombinant LVGFP-rGAD65 vector was successfully constructed.rLV-rGAD65-infected primary fibroblasts in vitro and the expressed rGAD65 catalyzed the formation of GABA from glutamic acid.In vivo,the concentration of GABA in the SNr was increased after rLV-rGAD65 injection into the STN.
目的谷氨酸脱羧酶2 (glutamic acid decarboxylase 2,GAD65) 是γ-氨基丁酸(gamma-aminobutyric acid, GABA)的合成酶。本研究拟构建重组大鼠GAD65基因的慢病毒载体(recombinant lentivirus-rGAD65,rLV-rGAD65),并在体内外分析其功能。方法用RT-PCR法克隆大鼠GAD65基因的cDNA 并亚克隆至慢病毒载体上,形成重组慢病毒质粒(rLV-GFP-rGAD65)。在包装质粒的帮助下,获得重组慢病毒颗粒(rLV-rGAD65)并检测其滴度。用rLV-rGAD65感染原代培养的大鼠肺成纤维细胞,并用免疫细胞化学和蛋白印迹法检测rGAD65在成纤维细胞中的表达,用高效液相法(high-performance liquid chromatograph, HPLC)检测培养上清中GABA的含量。在体内, rLV-rGAD65 定点注射到Sprague-Dawley大鼠的丘脑底核(subthalamic nucleus,STN)。用免疫组织化学和蛋白印迹法检测GAD65基因在STN中的表达水平,HPLC检测黑质网状部(substantia nigra pars reticulata,SNr) 中GABA的含量。结果 rGAD65 的序列与GenBank几乎一致,没有碱基的突变。rLV-rGAD65的滴度达6.8 × 108/mL。在体外,rLV-rGAD65对成纤维细胞的感染效率可达80%,而对照组中几乎没有GAD65蛋白的表达。在上清液中,GABA的含量达到了(48.14 ± 9.35) nmol/L。在体内, rGAD65与绿色荧光蛋白共表达于注射侧的STN区,GAD65蛋白的含量明显高于对照组和注射的对侧。GABA在SNr 中的含量也从(5.95 ± 1.09) 升高到(12.44 ± 3.79) nmol/g。结论重组GAD65载体构建成功。在体外,重组慢病毒颗粒可以感染原代培养的成纤维细胞,并能催化GABA的合成。在体内,重组慢病毒颗粒可以感染STN的神经元并能使SNr中GABA的含量增加。
