脂肪储存小滴蛋白5基因真核表达载体的构建及亚细胞定位  

Construction of eukaryotic fusion expression vectors of LSDP5 and cellular localization

作　　者：刘芳[1,2] 张利军[1] 叶菁[1] 张丽英[1] 李烦繁[1] 李航[1] 谷雨[1] 李青[1] 

机构地区：[1]第四军医大学西京医院病理科,陕西西安710032 [2]解放军第518医院,陕西西安710043

出　　处：《细胞与分子免疫学杂志》2010年第5期438-439,443,共3页Chinese Journal of Cellular and Molecular Immunology

基　　金：国家自然科学基金资助项目(30671087;30700268)

摘　　要：目的:构建pCMV5-HA与脂肪储存小滴蛋白5(LS-DP5)的融合基因真核表达载体,观察其在非洲绿猴肾成纤维细胞COS7中的表达,通过免疫荧光技术确定LSDP5的亚细胞定位。方法:采用NdeI和BamHI酶切pCMV5-HA质粒,用BglII和NdeI酶切pMD18-LSDP5质粒,克隆到pCMV5-HA载体,测序鉴定后,通过脂质体法转染COS7细胞,通过免疫荧光技术观察其在COS7细胞中的表达,并利用荧光染料Bodipy493/503定位脂滴,探讨LSDP5与脂滴之间的关系。结果:酶切及DNA测序证实重组质粒pCMV5-HA-LSDP5构建成功。荧光显微镜观察显示,pCMV5-HA-LSDP5融合蛋白定位于细胞质内,并定位于脂滴表面。结论:成功构建了重组质粒pCMV5-HA-LSDP5;融合蛋白分布于细胞质并与脂滴存在共定位关系。AIM：To construct the eukaryotic fusion expression vectors and localization of （lipid storage droplet protein 5,LSDP5） in COS7 cells.METHODS：Eukaryotic expression plasmid pCMV5-HA was digested with Nde I and BamH I and pMD18-LSDP5 plasmid was digested with Bgl II and Nde I.Full length LSDP5 was subcloned into pCMV5-HA.After confirmed by restriction enzyme digestion analysis and sequencing,the plasmid pCMV5-HA-LSDP5 was transfected into COS7 cells using Lipofectamine 2000 and the expression and localization of the fusion protein was detected by fluorescent microscope.RESULTS：Restriction digestions and sequencing assays showed that the recombinant plasmid of pCMV5-HA-LSDP5 was successfully constructed and the fusion proteins were detected in cytoplasm and were targeted to the surfaces of lipid droplets visualized by Bodipy 493/503 in COS7 cells.CONCLUSION：The recombined plasmid pCMV5-HA-LSDP5 expressing the fusion protein of LSDP5 and HA-tagged is successfully constructed and the fusion protein distributes in cytoplasm and is co-localized with lipid droplets.

关 键 词：脂肪储存小滴蛋白5 融合蛋白 pCMV5-HA COS7细胞 亚细胞定位 

分 类 号：R392.12[医药卫生—免疫学]