基于FMDV IRES的双顺反子载体的构建及体外表达分析  

Construction and Expression Analyzing of Bicistronic Vector Based IERS From FMDV

作　　者：郑海学[1] 郭慧琛[1] 靳野[1] 刘湘涛[1] 谢庆阁[1] 

机构地区：[1]中国农业科学院兰州兽医研究所家畜疫病病原生物学国家重点实验室农业部畜禽病毒学重点开放实验室

出　　处：《中国生物工程杂志》2007年第8期29-33,共5页China Biotechnology

基　　金：国家"973"计划资助项目(2005CB523201);国家科技支撑计划资助项目(2006BAD06A03)

摘　　要：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20～48h,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。The internal ribosome entry site （IRES） element from FMDV was amplified by RT-PCR, and was eloned into peDNA3.1 （ ＋ ） veetor, resulted in a bieistronie veetor. To determine whether the bieistronic mRNAs from the veetor ean be translated, the enhaneed green fluoreseent protein （EGFP） gene was eloned into the downstream of the IERS element, then BHK-21 eells was transfeeted by the reeombinant plasmid. The green fluoreseenee in the transfeeted eells was observed under a fluoreseenee mieroseope equipped with a video doeumentation system. And the expressional effieieney was analyzed with flow eytometry （ FCM ）. The results show that the IRES element from FMDV has the role of initiating CAP-independent translation ,and lay foundation for researching funetion of the element and interrelated proteins. It would be potential for expressing target gene by the bieistronie veetor.

关 键 词：口蹄疫病毒 IRES 双顺反子 双顺反子载体 

分 类 号：Q78[生物学—分子生物学]