小鼠FABP4基因启动子驱动红色荧光蛋白载体的构建及在牛体细胞中的表达  

Construction of Red Fluorescent Protein Driven by Mouse FABP4 Promoter and Expression in Bovine Cells

作　　者：岳永莉[1] 于海泉[1] 

机构地区：[1]内蒙古大学哺乳动物生殖生物学及生物技术教育部重点实验室,内蒙古呼和浩特010021

出　　处：《华北农学报》2016年第6期26-30,共5页Acta Agriculturae Boreali-Sinica

基　　金：转基因生物新品种培育科技重大专项(2014ZX08010005)

摘　　要：小鼠脂肪型脂肪酸结合蛋白(FABP4)的启动子/增强子元件是脂肪组织特异性启动元件,为了鉴定该元件在牛体细胞中的启动效果,首先以小鼠肝脏DNA为模板,通过PCR克隆得到5.9 kb的FABP4基因启动子片段,连入p MD19-T载体后进行酶切及测序鉴定,经Eco T 22I酶切去除非核心启动子区后精简为2.3 kb的片段,通过SacⅡ酶切将5.9 kb片段和2.3 kb片段的启动子连入红色荧光蛋白载体p Ds-Red 2-1的多克隆位点,构建为表达质粒p MF5.9-Red和p MF2.3-Red,利用脂质体法将上述载体分别转染牛脂肪间充质干细胞及牛胎儿成纤维细胞,24 h后实时定量PCR检测红色荧光蛋白转录水平。结果表明,克隆得到的5.9 kb小鼠FABP4启动子片段酶切及测序结果正确,与红色荧光蛋白载体相连的载体p MF5.9-Red和p MF2.3-Red酶切结果与预期相符,表达载体构建成功,实时定量PCR结果显示以上2种细胞在转染后24 h红色荧光蛋白均有表达,且p MF2.3-Red的转录水平是p MF5.9-Red的2倍以上。成功构建了小鼠FABP4启动子驱动红色荧光蛋白表达载体p MF5.9-Red和p MF2.3-Red,5.9 kb片段和2.3 kb片段均可驱动外源基因在牛体细胞中转录,且2.3 kb片段启动效率高于5.9 kb片段。The promoter/enhancer cassette of mouse adipocyte-type fatty acid binding protein（FABP4） was widely used as the adipocyte specific element.To detect the effect of m FABP4 promoter on gene expression in bovine cells,5.9 kb m FABP4 promoter fragment was cloned from mouse liver genome and identified by digestion and sequencing after ligated to p MD19-T vector.By digestion of enzyme Eco T 22 I the non-core promoter fragment was deleted from the 5.9 kb fragment and another 2.3 kb core promoter fragment was obtained.The 5.9 kb and 2.3 kb fragment were inserted into the multiple cloning site of plasmid p Ds-Red 2-1 by enzyme Sac Ⅱ,respectively.As a result the vectors,p MF5.9-Red and p MF2.3-Red,were constructed successfully.By lipofection,the constructed p MF5.9-Red and p MF2.3-Red vectors were transfected into bovine adipose-derived stem cells and bovine embryo fibroblast cells,respectively.24 h after transfection,Real-time PCR was applied to detect the transcription of red fluorescent protein in transfected cells.Results showed that the sequence of cloned 5.9 kb fragment was correct,and the 5.9 kb and the 2.3 kb fragments were correctly ligated into vectors.The mRNA of red fluorescent protein was both detected in transfected bovine adipose-derived stem cells and bovine embryo fibroblast cells,and the expression level displayed 2 fold higher in p MF2.3-Red than that of p MF5.9-Red.In conclusion,this study constructed the ex-pression vector p MF5.9-Red and p MF2.3-Red with 5.9 kb or 2.3 kb fragment of mouse FABP4 gene as promoter region could initiate the transcription of foreign genes in bovine cells and the transcription efficiency by 2.3 kb promoter fragment was higher than that of 5.9 kb promoter fragment.

关 键 词：小鼠FABP4启动子 载体构建 牛体细胞 

分 类 号：Q782[生物学—分子生物学]