【摘要】 目的　研究人芳香族左旋氨基酸脱羧酶 (AADC)基因在多巴胺代谢过程中的作用。方法　从人胎儿黑质组织中提取总RNA ,以RT PCR法扩增AADCcDNA片段 ,克隆于 pGEM T载体中。测序正确后再构建真核表达载体 ,转染猴肾成纤维细胞COS 7,原位杂交检测AADC表达 ,加入L DOPA后 ,用高效液湘色谱测定转AADC细胞的多巴胺(DA)生成量 ,以了解转基因细胞AADC基因的功能活性。结果　RT PCR扩增出 144 2bp的cDNA片段与Genbank[NM 0 0 0 790 ]登录的序列相同 ,构建真核表达载体 pBK RSV AADC ,原位杂交证实其在COS 7表达阳性率为 70 % - 80 % ,在AADC活性作用下实验组的DA生成量是对照组的 4倍以上 (P <0 0 1)。结论　所克隆的AADC基因可在真核细胞中表达并具有生物活性。可望用于帕金森病的基因治疗。更多还原

【Abstract】 ? Objective To investigate the role of human aromatic amino acid decarboxylase (AADC) in the metabolism of dopamine (DA). Method The total RNA was isolatedfrom fetal brain. RT-PCR was performed by using special primersof AADC. The cDNA fragment of AADC was ligated into plasmid pGEM-T. An expression vector pBK-RSV-AADC was constructed and then transfected into COS-7 cells. Result The amplified fragment was 1442bp with RT-PCR, and the sequence was exactly the same as that reported from Genbank \. The expression of the gene in the transfected COS- 7 cells wasassayed by in situ hybridazation and the ratio of positive cells was 70～80%. The activity was evaluated by HPLC, and the COS 7 cells transfected with AADC produced 4 times as much DA as those cells without transfection. Conclusion The cloned human AADCgene can be expressed in COS-7 cells and show bioactivity. It could be used for the gene therapy of Parkinson’s disease. 〔更多还原