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

  • 摘要: 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。

     

    Abstract: 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. Conclusions: These findings suggest that THmRNA is a newly and valuable tool to the treatment for Parkinson's disease.

     

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