真核表达的EF-1α-Flag蛋白对体外蛋白翻译的影响

Effect of EF-1α-Flag Protein Expressed in Eukaryotic Cell on Protein Translation in vitro

目的:构建EF-1α-Flag到pcDNA3.0表达载体上,在哺乳动物细胞中表达并纯化延伸因子1α(EF-1α),测定其对体外蛋白翻译的影响。方法:通过PCR技术从293T细胞cDNA文库中扩增EF-1α基因片段,连接到pcDNA3.0载体上,经电泳、测序和转入细胞中检测EF-1α-Flag蛋白表达等方式验证所构建的重组质粒是否正确,然后经Flag肽置换法纯化出EF-1α蛋白用于体外蛋白翻译实验,萤光素酶活性检测翻译出的蛋白含量。结果:测序和蛋白表达鉴定结果表明扩增的EF-1α-Flag基因序列正确,所构建的重组载体在哺乳动物细胞中能获得表达;考马斯亮蓝染色发现经Flag肽置换的EF-1α蛋白纯度较高,在体外蛋白翻译实验中EF-1α蛋白能促进蛋白的翻译。结论:从哺乳动物细胞中纯化的EF-1α蛋白能促进蛋白翻译,可能与其作为翻译延伸因子相关,为进一步研究EF-1α的功能奠定了基础。

Objective： Using in vitro translation assay to determine the role of elongation factor 1α（EF-1α） inprotein translation. Methods： The EF-1α gene was amplified from the 293 T cell cDNA library and was cloned in-to the pcDNA3.0 vector. The recombinant plasmid was verified by the agarose electrophpresis analysis and sequenc-ing. Further, the Flag-tagged EF-1α protein was expressed in mammalian cells, precipitated with anti-Flag aga-rose beads, and eluted with Flag peptide. The in vitro translation assay was performed and the luciferase activitywas measured. Results： The results of sequencing and expression demonstrated that the EF-1α gene has been cor-rectly cloned and could be expressed in mammalian cells. Using SDS-polyacrylamide gel elctrophoresis and Coo-massie brilliant blue staining, high purity EF-1α protein eluted by Flag-peptide was identified. The data of in vitro translation assay suggest that the purified EF-1α protein can induce protein translation in vitro. Conclusion：The EF-1α expressed in mammalian cells and crude eluted could be used in in vitro translation assay and pro-mote protein translation in vitro, which may be associated with its function as a translation elongation factor.