人激活转录因子5基因真核表达载体的构建及其生物学功能研究

Construction of myc-Tagged Human Activating Transcription Factor 5 Eukaryotic Expressionvector and its Activity Detection

目的:构建带myc标签的人激活转录因子5(ATF5)的真核表达载体,获得myc-ATF5融合蛋白,并对其生物学功能进行初步检测。方法:以本实验室保存的带有XL5标签的ATF5质粒为模板,采用PCR技术扩增ATF5编码序列,将其插入pXJ-40-myc载体中,Western印迹检测其表达;将重组质粒与空载体分别转染人胚肾293T细胞,通过Western印迹和qRT-PCR检测其下游基因哺乳动物雷帕霉素靶蛋白(mTOR)在蛋白和mRNA水平的变化。结果:双酶切和测序结果表明myc-ATF5真核表达质粒构建成功,转染293T细胞后获得表达;Western印迹和qRT-PCR证明myc-ATF5可升高mTOR的转录和蛋白水平。结论:构建了带myc标签的人ATF5真核表达载体,为进一步研究ATF5在自噬中的功能奠定了基础。

Objective： To construct the eukaryotic expression vector of human activating transcription factor 5 （ATF5） labeled with myc tag and detect its activity. Methods： Human ATF5 gene was obtained from XL5-ATF5 plasmid by PCR and cloned into myc vector. Human 293T cells were transfected with the recombinant plasmid myc-ATF5 and the expression was detected by Western blot. The mRNA and protein level of mechanistic target of rapamycin（mTOR） gene, which was supposed to be upregulated by myc-ATF5, was identified by Western blot and qRT-PCR. Results： ATF5 eukaryotic expression vector labeled with myc tag was successfully constructed by double digestion identification. The inserted fragment was confirmed to be correct by sequencing. Human ATF5 pro tein was expressed in human 293T cells as shown by Western blot. In addition, both qRT-PCR and Western blot results showed that human ATF5 protein could up-regulate the protein and mRNA levels of mTOR. Conclusion： The eukaryotic expression vector of myc-ATF5 was successfully constructed and expressed in human 293T cells, and it could up-regulate the protein and mRNA levels of mTOR, which had laid foundation for the further study of the role of ATF5 in autophagy.