摘要
选取由核表达的线粒体蛋白细胞色素C氧化亚单位Ⅷ(COX8)的前导序列为靶序列,从人胚胎肺成纤维细胞中扩增出COX8的前导序列,插入到pcDNA3.1/myc-HisA中,并将pDsRED1-n1中的红色荧光蛋白序列RFP克隆至COX8的下游,形成融合蛋白。在脂质体的介导下,将重组载体转染至肿瘤细胞中,在荧光显微镜下观察其在细胞内的表达及分布情况。构建的靶向线粒体的载体以及以红色荧光蛋白为报告基因的靶向线粒体的载体,经酶切、DNA序列测定,结果表明构建正确。将pcDNAmito-RFP转染到HeLa细胞的线粒体中,16h即可见散在荧光,72h达高峰,第10d开始减弱。以上结果表明成功构建了以红色荧光蛋白为报告基因的线粒体靶向的特异表达载体,在靶序列的引导下将红色荧光蛋白输入到线粒体中,为进一步对线粒体疾病的基因治疗研究提供了重要工具。
To construct a expression vector targeting mitochondrial with red fluorescent protein. The sequence of human cytochrome C oxidase subunit Ⅷ （COX8） was selected as leader sequence that was amplified using PCR technique from human embryo lung fibroblast cells, then cloned to pcDNA.3.1/myc-HisA, the RFP gene from pDsRED1-n1 vector was inserted to C terminus of the leader sequence. The plasmid was transfected into HeLa cells with LipofectAMINE 2000. The location and expression of the pcDNAmito-RFP was observed with fluorescence microscope. The recombinant vector was verified correctly by enzyme digestion, PCR and DNA sequence analysis. The pcDNAmito-RFP vector was highly expressed in the mitochondrial of HeLa cells. The red fluorescence was oppeared as early as 16 h, strongest at 72 h after transfection. The mitochondrial targeting expression vector that containing RFP reporter has been constructed successfully, it provides an important and convenient tool to study on therapy of mitochondrial disease.
出处
《生物技术通讯》
CAS
2005年第4期371-373,共3页
Letters in Biotechnology
基金
国家自然科学基金项目(30200141)
关键词
线粒体
表达载体
靶向
红色荧光蛋白
mitochondrial
expression vector
target
red fluorescent protein
