[1]雒喜忠,王阁,郑继军,等.RAI16基因真核表达载体的构建及在HepG2中的表达[J].第三军医大学学报,2009,31(17):1620-1624.
 LUO Xi-zhong,WANG Ge,ZHENG Ji-jun,et al.Construction of eukaryotic expression vector for RAI16 and its expression in HepG2 cells[J].J Third Mil Med Univ,2009,31(17):1620-1624.
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RAI16基因真核表达载体的构建及在HepG2中的表达(/HTML )
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《第三军医大学学报》[ISSN:1000-5404/CN:51-1095/R]

卷:
31卷
期数:
2009年第17期
页码:
1620-1624
栏目:
论著
出版日期:
2009-09-15

文章信息/Info

Title:
Construction of eukaryotic expression vector for RAI16 and its expression in HepG2 cells
作者:
雒喜忠王阁郑继军陈川张志敏李琼许文胡庆王东李增鹏
第三军医大学大坪医院野战外科研究所肿瘤中心
Author(s):
LUO Xi-zhong WANG Ge ZHENG Ji-jun CHEN Chuan ZHANG Zhi-min LI Qiong XU Wen HU Qing WANG Dong LI Zeng-peng
Cancer Center, Institute of Surgery Research, Daping Hospital, Third Military Medical University, Chongqing 400042, China
关键词:
RAI16基因融合蛋白增强型绿色荧光蛋白HepG2细胞
Keywords:
homo sapiens retinoic acid induced 16 generecombinant fusion proteinEGFPHepG2 cell
分类号:
R341;R394-33; R735.7
文献标志码:
A
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 green fluorescent 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。
Abstract:
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-C1 vector to construct the recombinant eukaryotic expression vector pEGFP-C1-RAI16. The expression of the fusion protein RAI16-EGFP was detected after the transfection of pEGFP-C1-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-C1, 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 transfected 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-C1-RAI16 eukaryotic expression vector is successfully constructed, and the fusion protein RAI16-EGFP is expressed in the HepG2 cells after transfection.

参考文献/References:

雒喜忠, 王阁, 郑继军, 等.RAI16基因真核表达载体的构建及在HepG2中的表达[J]. 第三军医大学学报,2009,31(17):1620-1624.

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更新日期/Last Update: 2009-09-14