大鼠TH基因mRNA的合成及其在真核细胞中的表达

Cloning, synthesizing and eukaryotic expression of rat TH gene mRNA

目的 :克隆大鼠酪氨酸羟化酶 (tyrosinehydroxylase,TH)基因的核心cDNA序列 ,并在体外合成THmR NA ,观察其在真核细胞中的表达情况。方法 :采用大鼠黒质细胞cDNA为模板 ,设计特定的引物进行PCR扩增并构建真核表达载体pcDNA 3.1/TH ,在体外转录后获得THmRNA。将获得的THmRNA转染到体外培养的NIH 3T3细胞内 ,用免疫组织化学方法和免疫印迹方法检测其表达。结果 :免疫组织化学方法和免疫印迹方法进行检测后显示转染的THmRNA可以在NIH 3T3细胞内表达TH ,时间达到两周。结论 :采用THmRNA对帕金森病进行的基因治疗可能是一条新的和有价值的途径。

Objective: To obtain the core cDNA sequence of rat tyrosine hydroxylase(TH) and synthesize THmRNA in vitro. To observe the expression of THmRNA in eukaryotic cell. Methods: The fulllength of cDNA from rat TH was used as template and the specific oilgonucleotide primers were designed for Polymerase Chain Reaction(PCR). An eukaryotic vector, plasmid pcDNA3.1/TH, was constructed, then Synthesizing THmRNA in vitro. The newly Synthesized THmRNA was transfected in vitro to the cultured NIH3T3 cells. The expression of TH was tested by immunocytochemistry and Westernblot. Results: The expression of TH was shown in NIH3T3 by immunocytochemistry staining and Westernblot. The transfected THmRNA can express TH in cultured NIH3T3 cells as long as 2 weeks. Conclusion: These findings suggest that THmRNA is a newly and valuable tool to the treatment for Parkinson's disease.