带组氨酸标签的癌蛋白SET真核表达载体的构建及其表达  

Construction of the Eukaryotic Expression Vector of SET with Six Hisditine Tag and Its Expression in L-02 Cell Lines

作　　者：陈谋通[1,2] 刘建军[2] 杨汝德[1] 何浩伟[2] 蒋英芝[2] 贺连华[2] 

机构地区：[1]华南理工大学生物科学与工程学院,广州510006 [2]深圳市疾病预防控制中心毒理研究室,深圳518020

出　　处：《热带医学杂志》2009年第5期480-482,479,共4页Journal of Tropical Medicine

基　　金：国家自然科学基金(No.30571557);深圳市科技计划重点项目(No.200801010)

摘　　要：目的为研究三氯乙烯诱导的差异蛋白SET在肝细胞L-02中的相互作用,构建了癌蛋白SET和His标签融合表达的真核表达载体pcDNA3.1(+)/SET-His。方法从L-02肝细胞中提取总RNA,采用RT-PCR扩增SET-His基因并进行双酶切,序列纯化后定向克隆至pcDNA3.1/zeo(+)载体,阳性克隆载体进行双酶切和测序鉴定,阳性克隆载体瞬时转染入L-02肝细胞,利用Western blotting检测SET-His融合蛋白的表达。结果利用RT-PCR从L-02细胞总RNA中成功克隆出SET基因,经双酶切和测序鉴定证实pcDNA3.1(+)/SET-His真核表达载体构建成功。经Western blotting验证表明,SET-His融合蛋白在肝细胞中获得高效表达。结论该结果为研究SET蛋白相互作用以及三氯乙烯致机体损伤的机理奠定了基础。Objective To construct the vector pcDNA3.1/SET-His for the expression of differential protein SET in L-02 liver cells, which can be used for further study of molecular mechanism of SET. Methods Total RNA was extracted from human liver cells, the open reading frame of SET was obtained by RT-PCR, and then the recombinant vector pcDNA3.1/SET-His were constructed and transformed into E.coli DH 5α. After the sequence was confirmed by using double enzyme digestion and sequencing, L-02 liver cells were transfected with this recombinant vector using Lipofeetamine 2000. Western blotting was used to detect the expression of SET-His fusion protein. Results The oneogene SET was successfully cloned. Results from double enzyme digestion and sequencing showed that the fusion gene （SET- His） were correctly inserted into the vector pcDNA3.1/zeo （＋）. The peDNA3.1/SET-His can transcript and express the fusion protein SET-His efficiently in L-02 liver cells verified by western blotting. Conclusion The recombinant vector pcDNA3.1/SET-His was successfully constructed, which lays the foundation for further studying the protein- protein interactions of oncoprotein SET in L-02 liver cells and the pathogenic mechanism causing by trichloroethylene.

关 键 词：三氯乙烯 癌蛋白SET 组氨酸标签 融合表达 

分 类 号：Q51[生物学—生物化学]