真核表达载体pEGFP-VASP-lC的构建及其在HeLa细胞中的表达  

Construction of Eukaryotic Expression Vector pEGFP-VASP-lC and Its Expression in HeLa Cells

作　　者：张利军[1] 魏蕾[1] 王婷婷[1] 张京伟[2] 彭小春[1] 文显梅[1] 李华[1] 

机构地区：[1]武汉大学医学院病理生理学教研室,武汉430071 [2]武汉大学中南医院肿瘤外科,武汉430071

出　　处：《武汉大学学报（医学版）》2006年第4期421-424,F0002,共5页Medical Journal of Wuhan University

基　　金：教育部留学归国人员科研基金项目(编号:301170272)

摘　　要：目的:构建真核表达载体pEGFP-VASP-lC并稳定转染人宫颈癌细胞系HeLa细胞,为进一步探讨VASP-lC区介导的VASP的寡聚化在HeLa细胞迁移中的作用奠定基础。方法:用RT-PCR技术从人内皮细胞株ECV304细胞中获得VASP-lC区的cDNA并将其插入表达载体pEGFP-C1中,构建的重组质粒经脂质体介导转染HeLa细胞,Western blot鉴定VASP-lC区在HeLa细胞中的稳定表达。结果:RT-PCR成功地扩增出一条约310bp的片段,经限制性内切酶酶切图谱分析和DNA序列测定证实目的基因已插入重组质粒,荧光显微镜下观察到稳定转染的细胞发出较强绿色荧光,Western blot证明人VASP-lC区能以融合蛋白的形式在HeLa细胞中稳定表达。结论:成功构建了真核表达载体pEGFP-VASP-lC,获得了稳定表达人VASP-lC区基因的HeLa细胞克隆,便于进一步探讨VASP-lC区介导的VASP的寡聚化在HeLa细胞迁移中的作用。Objective： To construct eukaryotic expression vector pEGFP-VASP-lC and obtain positive HeLa cell clones expressing lC region of human VASP stably, and to establish a cell model for exploring the role of lC region-mediated oligomerization of VASP in the the migration of HeLa cells further. Methods： The cDNA encoding the lC region of human VASP was amplified by RTPCR from the total RNA isolated from the human ECV304 cells and was inserted into pEGFP-C1 vector to construct the recombinant eukaryotic expression vector pEGFP-VASP-lC. The recombinant plasmid was then transferred into HeLa cells by liposome. Overexpression of the lC region of human VASP in the transfected HeLa cells was confirmed with Western blot. Results： By the use of RT-PCR, a 310 bp DNA fragment was successfully amplified from the human ECV304 cells, which matched the length of cDNA encoding the lC region of human VASP. Enzyme digestion analysis and DNA sequencing showed that the target gene was cloned into recombinant vector. Green fluorescence was emitted from transfected cells under fluorescent microscope. Western blot analysis revealed the fusion protein EGFP-VASP-lC could be expressed stably in the transfected HeLa cells. Conclusion： The eukaryotic expression pEGFP-VASP-lC was successfully constructed. The positive HeLa cell clones expressing the lC region of human VASP stably were obtained, which may be a cell model for studying the role of lC region-mediated oligomerization of VASP in the migration of HeLa cell further.

关 键 词：血管扩张刺激磷蛋白 真核表达载体 HELA细胞 表达 

分 类 号：R730.23[医药卫生—肿瘤]