人三磷酸鸟苷酸环化水解酶Ⅰ基因的克隆与表达

CLONING AND EXPRESSION OF HUMAN GTP CYCLOHYDROLASEⅠ(GCHⅠ)

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 The cDNA of human guanosine triphosphate(GTP) cyclohydrolase(GCHⅠ) was cloned in order to investigate the roles of GCHⅠ in metabolism of dopamine(DA). Methods After the total RNA was isolated,the RT\|PCR of GCHⅠ was performed by using specially designed primers.The fragment of GCHⅠ was ligated into plasmid of pGEM\|T\|easy and sequence was performed.An expression vector pCI\|neo\|GCHⅠ was reconstructed and then transfected into the cell line of COS7. Results The GCHⅠ gene sequence was exactly the same as what reported in the GeneBank.Expression of the gene in transfected COS7 cells was assayed by using in situ hybridization and the rate of positive cells was 70%\|80%.Conclusion\ The results showed that the cloned human GCHⅠ in present study might be used for gene therapy of Parkinson disease(PD).[