双效表达载体的构建及其U6启动子的功能效率鉴定  

Construction of Double-effect Vector and Function Identification of U6 Promoter

作　　者：项海涛[1,2] 杨彬 温峰琴[1] 胡永浩[1] 柳纪省[2] 

机构地区：[1]甘肃农业大学动物医学院,兰州730070 [2]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,农业部草食动物疫病重点开放实验室,兰州730046

出　　处：《生物技术通报》2009年第4期82-85,90,共5页Biotechnology Bulletin

基　　金：国家"973"前期专项(2004CCA00500);国家"863"项目(2006AA10A204)

摘　　要：利用pBudcE4.1双表达载体构建shRNA与蛋白共表达载体,为双效疫苗的研制提供新的研究思路。以含U6启动子的载体为模板,PCR扩增得到U6启动子,用其置换载体pBudcE4.1内的CMV启动子的核心部分构建shRNA与蛋白共表达载体。用干扰绿色荧光蛋白表达的方法鉴定重组载体中的U6启动子能否启动shRNA的表达。经PCR扩增、双酶切鉴定及DNA测序证明成功构建了载体pBudcE4.1-U6。用干扰载体pBudcE4.1-U6-eGFPshRNA与含eGFP的载体共转染293T细胞后,荧光显微镜观察显示eGFP的表达量下降;流式细胞仪检测细胞的转染效率降低。研究结果证明U6启动子正常发挥作用。成功构建RNAi与蛋白共表达载体,为利用该载体研制动物双效疫苗奠定了基础。The co-expression vector pBudcE4.1 was used to construct to express shRNA and protein simultaneously. U6 promoter was amplified by PCR using plasmid containing U6 promoter as template, and the core section of CMV promoter of pBudcE4. 1 was substituted for U6 promoter. The plasmid pBudcE4, 1-U6 was constructed. Interfering of expression of EGFP was used to identify whether the shRNA can be promoted by U6 promoter. Results showed that the plasmid pBudcE4, 1-U6 was proved successfully constructed identified with PCR,restriction endonuclease analysis and DNA sequence analysis. The expression of green fluorescent protein was reduced that was observed by fluorescence microscopy. Flow cytometry detection exhibited that transfection efficiency decreased. The results also indicated that the U6 promoter develop its normal function. Thus the vector which co-express shRNA and protein would be used to manufacture double-effect vaccine.

关 键 词：RNAI 共表达载体 U6启动子 构建 鉴定 

分 类 号：Q78[生物学—分子生物学] S572[农业科学—烟草工业]