CRT-E2-EGFP表达、胞内定位及其对B16细胞凋亡的影响  

Expression and functional analysis of CRT-E2-EGFP fusion protein in B16 cells

作　　者：杨建林[1] 柳长柏[1] 李丹莉[1] 韩莉[1] 

机构地区：[1]三峡大学分子生物学研究所,湖北宜昌443002

出　　处：《细胞与分子免疫学杂志》2009年第10期897-899,共3页Chinese Journal of Cellular and Molecular Immunology

基　　金：湖北省教育厅自然科学研究重点项目(D200713004)

摘　　要：目的:观察CRT-E2融合蛋白在小鼠黑色素瘤B16细胞中的表达及其定位,并探讨CRT引起的E2蛋白表达定位改变对B16细胞增殖及调亡的影响。方法:构建荧光真核表达载体pEGFP-CRT、pEGFP-E2、pEGFP-CRT-E2,将上述载体以及空载体pEGFP-C1经脂质体瞬时转染B16细胞,West-ernblot分析确认目的蛋白表达。荧光显微镜下观察CRT-E2融合蛋白在细胞中表达定位;流式细胞术检测不同重组表达载体转染B16细胞24h、48h后,对细胞周期的影响和诱导凋亡的情况。结果:CRT-E2融合蛋白在B16细胞中正确表达,主要分布于细胞质,能将细胞阻滞于G1期,有效诱导肿瘤细胞发生凋亡(P<0.01)。结论:成功构建CRT-E2真核表达载体,外源性CRT能够引起E2蛋白表达定位于细胞质,并明显促进了肿瘤细胞的调亡。AIM： To detect the expression of the Calreticulin and HPV E2 Fusion Protein in B16, and study the effects on proliferation and apoptosis of BI6 cell lines in vivo. METHODS： To construct eukaryotic fluoresce expression vector pEGFP-CRT-E2, pEGFP-CRT and pEGFP-E2. Then the recombinant plasmids were transfected into B16 cells by Lipofectamine 2000. The expression of proteins was detected by Western blot. The location of different GFP fusion proteins in B16 was tested by inverted fluoresce microscope. Flow cytometry was applied to detect the effects of fusion proteins on the growing of B16 and then the apoptosis effects of B16 induced by different proteins were observed. RESULTS： The correctly constructed recombinant plasmid pEGFP-CRT-E2 and the expression of CRT-E2 gene could be detected in B16 cells. Apoptosis of B16 cells could be detected after the transient transfection. Meanwhile, the apoptosis rate of B16 cells transfected by pEGFP-CRT-E2 was much higher than that of control cells. And cell cycle G1/G0 arrest was also found （ P 〈 0.01 ）. CONCLUSION： The eukaryotic expression plasmid pEGFP-CRT-E2 is successfully constructed and it could correctly express the fusion protein in B16 cells. And the B16 cells transfected by plasmid pEG- FP-CRT-E2 could induce apoptosis.

关 键 词：HPV E2 钙网蛋白 凋亡 

分 类 号：R392.11[医药卫生—免疫学]