EGFP-CIDE-3及D sRed1-CIDE-3融合基因真核表达载体的构建及其在293T细胞中的表达和定位  

Construction of eukaryotic fusion expression vectors containing CIDE-3 with EGFP and DsRed1 and determination of their expressions and localization in 293T cells

作　　者：谷雨[1] 李青[1] 叶菁[1] 李烦繁[1] 闵婕[1] 张丽英[1] 马钰[1] 李航[1] 刘芳[1] 

机构地区：[1]第四军医大学西京医院病理科,陕西西安710032

出　　处：《医学研究生学报》2008年第9期911-914,I0002,共5页Journal of Medical Postgraduates

基　　金：国家自然科学基金资助项目(批准号:30671087)

摘　　要：目的:分别构建EGFP、DsRed1与CIDE-3的融合基因真核表达载体,观察其在人胚肾上皮细胞293T中的表达,确定CIDE-3的亚细胞定位。方法:分别以质粒pEGFP-C3、pDsRed1-N1及本实验室克隆得到的质粒pET28a(+)-CIDE-3为模板,PCR扩增EGFP、DsRed1 DNA片段及人CIDE-3基因的CDS序列,再分别将EGFP、CIDE-3及DsRed1、CIDE-3克隆入真核表达载体pShuttle-CMV中。酶切、测序鉴定后,经磷酸钙转染入293T细胞,通过荧光显微镜观察其在293T细胞中的表达,并利用荧光染料Bodipy 493/503定位脂滴,探讨CIDE-3与脂滴之间的关系。结果:酶切及DNA测序证实,重组质粒pShuttle-CMV-EGFP-CIDE-3和pShuttle-CMV-DsRed1-CIDE-3构建成功。荧光显微镜观察显示,pShuttle-CMV-EGFP-CIDE-3融合蛋白定位于细胞质,pShuttle-CMV-DsRed1-CIDE-3融合蛋白也定位于细胞质,并与脂滴存在共定位关系。结论:成功构建了重组质粒pShuttle-CMV-EGFP-CIDE-3和pShuttle-CMV-DsRed1-CIDE-3;两者均可在239T细胞中表达,融合蛋白分布于细胞质,并与脂滴存在共定位关系。Objective： To construct the eukaryotic fusion expression vectors of pShuttle-CMV-EGFPCIDE-3 and pShuttle-CMV-DsRed 1-CIDE-3 and to observe their expressions and localization in 293T cells. Methods ： The DNA segments of EGFP and DsRed 1 and the CDS of human CIDE-3 were respectively amplified from the plasmid pEGFP-C3, pDsRed l-N1 and pET28a （ ＋ ）-CIDE-3 by PCR. Then EGFP and CIDE-3 were subcloned into the vector pShuttle-CMV. DsRed 1 and CIDE-3 were also intro- duced to pShuttle-CMV. After confirmed by restriction enzyme digestion analysis and sequencing, these plasmids were transfected into 293T cells by Ca3 （ PO4 ） 2, and the expression and localization of the fusion protein were detected with the fluorescent microscope. Results： Restriction digestions and sequencing assays showed that the recombinant plasmids of pShuttle-CMV-EGFP-CIDE-3 and pShuttle-CMV-DsRed 1-CIDE-3 were successfully constructed and the fusion proteins were observed in the cytoplasm and co-localized with lipid droplets in 293T cells under the fluorescent microscope. Conclusion： The recombined plasmids of pShuttle-CMV-EGFP-CIDE-3 and pShuttle-CMV-DsRed 1-CIDE-3 were successfully constructed. The fusion proteins were distributed in the cytoplasm and co-localized with lipid droplets.

关 键 词：CIDE-3 融合蛋白 EGFP DsRed1 293T细胞 

分 类 号：Q782[生物学—分子生物学]