脑心肌炎病毒(EMCV)的内部核糖体进入位点活性的研究

Study on the Activity of EMCV IRES

脑心肌炎病毒(Encephalomyocarditis virus,EMCV)的内部核糖体进入位点(internal ribosomal entry site,IRES)已被广泛运用于蛋白的表达中,如应用于Clontech公司推出的pIRES2质粒对增强型绿色荧光蛋白(EGFP)的表达。但是由于对EMCV的IRES了解还不够深入而在利用EMCV的IRES进行基因表达过程中经常会遇到问题,很有必要对其进行更深入的研究。以红绿色荧光蛋白基因作为报告基因,通过分子克隆、荧光显微镜、荧光分光光度计、Western blot等分子细胞生物学手段,对EMCV的IRES末端序列与其活性的关系进行了深入研究。研究发现EMCV的IRES末端序列对其IRES的活性影响极大,而人们常用的报告基因EGFP本身也可能存在IRES。

The internal ribosomal entry site （IRES） from encephalomyocarditis virus （EMCV） was a popular element widely used in the plasmid to express proteins in eukaryotic ceils or cell-free extracts, such as the plRES2 plasmid developed by elontech. Although it had already been widely used, researchers often encountered in headache problems when manipulating it. Normally, the translation efficiency driving by the modified EMCV IRES was difficult to control. It＇ s necessary to study EMCV IRES mediated translation in detail. The relationship between the end sequence of EMCV IRES and its activity was determined. The red and green fluorescent protein genes were used as reporter genes, and ＂GCTAGA＂ bases were inserted at the end of EMCV IRES by molecular cloning. The expression efficiency was analyzed with fluorescence microscopy, fluorescence spectrophotometer, Western blot and other methods of molecular biology. As a result, it was found that additional bases at the end of EMCV IRES severely affected the IRES activity of EMCV, and the frequently used EGFP reporter gene may contain IRES element itself. These results indicate that investigators should pay more attention to the integrity of IRES sequences and their connection forms with the downstream sequence.