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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。
Purpose Glutamic acid decarboxylase 2 (GAD65) is a synthase of gamma-aminobutyric acid (GABA). In this study, recombinant lentivirus-rGAD65 (rLV-rGAD65) was constructed and its function was analyzed in vitro and in vivo. Methods The cDNA of rat GAD65 gene was cloned by RT-PCR and subcloned into lentiviral vector to form recombinant lentiviral plasmid (rLV-GFP-rGAD65). With the help of packaging plasmids, recombinant lentiviral particles (rLV-rGAD65) were obtained and their titers were determined. Primary cultured rat lung fibroblasts were infected with rLV-rGAD65 and the expression of rGAD65 in fibroblasts was detected by immunocytochemistry and Western blotting. The cells were detected by high-performance liquid chromatography (HPLC) GABA content in the supernatant. In vivo, rLV-rGAD65 was injected site-directed into the subthalamic nucleus (STN) of Sprague-Dawley rats. The expression of GAD65 gene in STN was detected by immunohistochemistry and Western blotting. The content of GABA in substantia nigra pars reticulata (SNr) was detected by HPLC. Results The sequence of rGAD65 was almost the same as that of GenBank. There was no base mutation. The titer of rLV-rGAD65 was 6.8 × 108 / mL. In vitro, rLV-rGAD65 infects fibroblasts up to 80% more efficiently, while there is almost no GAD65 protein expression in the control group. In the supernatant, the content of GABA reached (48.14 ± 9.35) nmol / L. In vivo, rGAD65 was co-expressed with GFP in the STN region of the injected side, and the content of GAD65 protein was significantly higher than that of the control group and contralateral side of injection. The content of GABA in SNr also increased from (5.95 ± 1.09) to (12.44 ± 3.79) nmol / g. Conclusion The recombinant GAD65 vector was successfully constructed. In vitro, recombinant lentivirus particles can infect primary cultured fibroblasts and can catalyze the synthesis of GABA. In vivo, recombinant lentiviral particles can infect STN neurons and increase the amount of GABA in SNr.