基于FMDV IRES的双顺反子载体的构建及体外表达分析

利用RT-PCR扩增出口蹄疫病毒小核糖体进入位点（IRES）序列，并定向克隆进pcDNA3.1(+)载体，构建成双顺反子真核表达载体。为了验证该载体是否能够转录出双顺反子mRNA，在IRES起始密码（ATG）下游正确插入增强型的绿色荧光蛋白基因（egfp），把重组质粒转染BHK-21细胞，培养20～48 h，在紫外显微镜下观察，能够看到典型的绿色荧光，表明载体能够体能够利用FMDV的IRES能够介导非帽依赖性表达外源基因。并通过流式细胞仪，与同样是CMV启动转录egfp的pGFPN1质粒在细胞中的表达的水平进行了比较。该载体的成功构建为体外表达双基因、双顺反子逆转录载体构建以及相关应用奠定基础，并有作为基因疫苗和标记定位基因治疗载体的潜力。

Construction and expression analyzing of bicistronic vector based IERS from FMDV

The internal ribosome entry site (IRES) element from FMDV was amplified by RT-PCR,and was cloned into pcDNA3.1( ) vector,resulted in a bicistronic vector.To determine whether the bicistronic mRNAs from the vector can be translated,the enhanced green fluorescent protein (EGFP) gene was cloned into the downstream of the IERS element,then BHK-21 cells was transfected by the recombinant plasmid.The green fluorescence in the transfected cells was observed under a fluorescence microscope equipped with a video documentation system.And the expressional efficiency was analyzed with flow cytometry (FCM).The results show that the IRES element from FMDV has the role of initiating CAP-independent translation,and lay foundation for researching function of the element and interrelated proteins.It would be potential for expressing target gene by the bicistronic vector.