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目的 研究人三磷酸鸟苷酸环化水解酶Ⅰ (GCHⅠ )基因在多巴胺代谢过程中的作用。 方法 从人胚胎肝脏中提取总RNA ,以RT PCR法扩增GCHⅠcDNA ,克隆于PGEM T easy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系COS7,原位杂交检测其表达。 结果 RT PCR扩增出 90 4bp的cDNA ,并成功构建真核表达载体pCI neo GCHⅠ ,原位杂交证实其在COS7表达阳性率为 70 %～ 80 %。 结论 GCHⅠ有望用于帕金森病的基因治疗。
Objective To study the role of GTP Ⅰ in dopamine metabolism. Methods Total RNA was extracted from the liver of human embryos, GCHⅠ cDNA was amplified by RT-PCR, cloned into PGEM T easy vector, and then sequenced. The eukaryotic expression vector was constructed and transfected into monkey kidney fibroblast cell line COS7 and detected by in situ hybridization Its expression. Results The 90 4bp cDNA was amplified by RT - PCR and the eukaryotic expression vector pCI neo GCH Ⅰ was successfully constructed. The positive rate of COS7 expression was 70% ~ 80% confirmed by in situ hybridization. Conclusion GCH Ⅰ is expected to be used for gene therapy of Parkinson’s disease.