【摘 要】
：
目的 为了探讨人谷氨酸脱羧酶(hGAD65)在PD治疗中的作用,拟克隆hGAD65基因,将其重组在腺相关病毒载体(rAAV)上,并在体外检测其功能,为体内实验积累数据.方法结果 取流产胚胎的脑组织(海马、皮质)应用RT-PCR的方法获得GAD65的cDNA,连于T-easy载体上.测序得到结果与genebank中的GAD65基因的一致率达到98％,几个突变点并没有产生错意突变.应用双酶切将GAD6
【机 构】
：
首都医科大学神经研究所 北京 100069;锦州医学院解剖学教研室,锦州121001 首都医科大学
【出 处】
：
中国神经科学学会第四次会员代表大会暨第七届全国学术会议(The 7th Biennial Meeting and the
论文部分内容阅读
目的 为了探讨人谷氨酸脱羧酶(hGAD65)在PD治疗中的作用,拟克隆hGAD65基因,将其重组在腺相关病毒载体(rAAV)上,并在体外检测其功能,为体内实验积累数据.方法结果 取流产胚胎的脑组织(海马、皮质)应用RT-PCR的方法获得GAD65的cDNA,连于T-easy载体上.测序得到结果与genebank中的GAD65基因的一致率达到98％,几个突变点并没有产生错意突变.应用双酶切将GAD65克隆入rAAV-CMV载体上,经酶切鉴定插入方向正确后,提取rAAV-GAD65、pAAV-RC和pHelper三个质粒.紫外分光光度计检测三个质粒的浓度,均达到1.0 μg/μL以上.应用无菌水调整三者浓度到1μg/μL,按比例共同转染HEK293细胞(包装rAAV-GAD65).转染72 h后,收集上清和细胞,经反复冻融(四次)裂解细胞,10 000 r/min离心10 min,收集上清,经0.45μm滤膜过滤,上清入Millipore柱子中进行浓缩.收集浓缩后的病毒上清液贮存于-80℃备用.取浓缩后的病毒上清液5 μL,稀释100倍,后依次稀释到1010倍,用梯度浓度的上清液转染HT1080细胞,检测病毒滴度.转染两天后,经免疫组化染色观察,rAAV-GAD65颗粒浓度达到了109 μg/mL.取106个病毒颗粒转染培养的人神经干细胞,48 h后,高压液相检测上清中的GABA含量.转染组与对照组相比具有显著性差异(P＜ 0.001),说明rAAV-GAD65具有将谷氨酸转变成GABA并释放到细胞外的能力.结论 本实验克隆所得的hGAD65具有一定的生物学功能,能用于在体的研究.
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