RAI16基因真核表达载体的构建及在HepG2中的表达  

Construction of eukaryotic expression vector for RAI16 and its expression in HepG2 cells

作　　者：雒喜忠[1] 王阁[1] 郑继军[1] 陈川[1] 张志敏[1] 李琼[1] 许文[1] 胡庆[1] 王东[1] 李增鹏[1] 

机构地区：[1]第三军医大学大坪医院野战外科研究所肿瘤中心,重庆400042

出　　处：《第三军医大学学报》2009年第17期1620-1624,共5页Journal of Third Military Medical University

基　　金：国家自然科学基金(30370341;30570410);教育部高等学校全国优秀博士学位论文作者专项基金(200261)~~

摘　　要：目的构建人视黄酸诱导蛋白16(retinoic acid induced 16,RAI16)与增强型绿色荧光蛋白(enhanced greenfluorescent protein,EGFP)的融合基因真核表达载体pEGFP-C1-RAI16,使RAI16-EGFP融合蛋白在人肝癌细胞株HepG2中得到表达。方法采用PCR技术,从含有目的基因的质粒克隆模板中钓取并扩增出RAI16全长编码基因,构建RAI16与EGFP的融合基因真核表达载体pEGFP-C1-RAI16,用脂质体转染技术将pEGFP-C1-RAI16导入HepG2,激光共聚焦分析RAI16亚细胞定位及Western blot检测RAI16-EGFP融合蛋白的表达。结果经转化细菌、抽提质粒、酶切鉴定和DNA序列分析证实,RAI16基因已正确插入pEGFP-C1中EGFP基因的下游,获得融合基因表达载体pEGFP-C1-RAI16。将pEGFP-C1-RAI16导入HepG2细胞24 h后,激光共聚焦分析显示RAI16-EGFP融合蛋白主要表达于细胞质内。Western blot结果显示,转染pEGFP-C1-RAI16 24、48 h和72 h后,RAI16基因在蛋白水平的表达逐渐增高,而AFP的表达逐渐下调。结论成功构建了真核表达载体pEGFP-C1-RAI16,并在HepG2细胞中进行了表达。Objective To construct the eukaryotic expression vector for homo sapiens retinoic acid induced 16 （RAI16） fused with the enhanced green fluorescent protein （EGFP） and explore the expression of the fusion protein RAI16-EGFP in HepG2 cells. Methods The intact RAI16 sequence was fished and amplified from the plasmid clone templates containing RAI16 sequence fragment by PCR. Then it was inserted into the pEGFP-Cl vector to construct the recombinant eukaryotic expression vector pEGFP-Cl -RAI16. The expression of the fusion protein RAI16-EGFP was detected after the transfection of pEGFP-Cl -RAI16 into the HepG2 cells. The subcellular localization of RAI16 and expression of RAI16-EGFP were observed by confocal fluorescence microscopy and Western blotting. Results The pEGFP-RAI16 recombinant expression vector, with RAI16 was exactly inserted in pEGFP-Cl , was constructed and confirmed by DNA sequencing, enzymatic digestion and PCR identification. In 24 h after the recombinant vector of fusion protein RAI16-EGFP was transfeeted into the cells, it was observed mainly in the cytoplasm. Western blot analysis showed the expression of RAI16 was up-regulated apparently at protein level after the cells were transfected with pEGFP-RAI16 for 24, 48 or 72 h. While, at the same time, the protein expression of AFP was decreased. Conclusion The pEGFP-CI-RAI16 eukaryotic expression vector is successfully constructed, and the fusion protein RAI16-EGFP is expressed in the HepG2 cells after transfection.

关 键 词：RAI16基因 融合蛋白 增强型绿色荧光蛋白 HEPG2细胞 

分 类 号：R341[医药卫生—基础医学] R394-33