广西巴马小型猪miR-181b慢病毒表达载体的构建和鉴定  

Construction and Identification of Lentiviral Vector with Guangxi Bama Mini-pig miR-181b

作　　者：吴敏[1] 黄建芳[1] 徐文文[1] 程晓芳[1] 郭晓萍[1] 杨秀荣[1] 郭亚芬[1] 兰干球[1] 

机构地区：[1]广西大学动物科学技术学院,南宁530004

出　　处：《基因组学与应用生物学》2016年第8期1944-1949,共6页Genomics and Applied Biology

基　　金：国家现代农业产业技术体系广西生猪创新团队项目(nycytxgxcxtd-03-15)资助

摘　　要：本试验旨在构建广西巴马小型猪miR-181b慢病毒表达载体,以广西巴马小型猪基因组DNA为模板,利用PCR扩增miR-181b前体序列,构建重组质粒p LV-miR-181b,经PCR和测序鉴定,阳性重组质粒转染C2C12细胞,荧光倒置显微镜下检测转染效率。结果显示,miR-181b前体序列长度为377 bp,与预期片段序列长度一致。将鉴定为阳性的miR-181b重组质粒转染C2C12细胞48 h后,在荧光显微镜下检测到较强的绿色荧光蛋白的表达,说明重组miR-181b质粒在C2C12细胞中具有较高的表达活性。本研究成功构建了具有高表达活性的miR-181b重组慢病毒表达载体,为研究miRNA-181b调控骨骼肌生长发育的功能机制提供实验基础。The purpose of this paper was to construct a recombinant lentiviral vector carrying of Guangxi Bama Mini-pig miR-181 b. The genomic DNA of Guangxi Bama Mini-pig was used as template to amplify pre-mir-181 b gene by PCR. Then recombinant plasmid of pLV-miR-18 l b was identified by transfecting into C2C 12 cells, obs- eying the transfection efficiency under fluorescence microscope. The results showed that 377 bp PCR amplified fragments were acquired, which was consistent with gene corresponding to the pre-miR-181b. In addition, the expression of C2C12 cells transfected with lentiviral vector was observed with high activity. Therefore, we succ- essfully constructed the lentiviral expression vector of pLV-miR-18 l b for miR-18 l b, and the experiment layed a good theoretical basis for the function and mechanism of miR-181 b, which might regulate skeletal muscle growth and development.

关 键 词：广西巴马小型猪 慢病毒载体 骨骼肌 miR-181b基因 

分 类 号：Q78[生物学—分子生物学]