[1]彭琬昕,孙瑶湘,陈琛,等.pDsRED-LC3真核表达载体的构建及其在肝癌细胞系HepG2中的表达和定位[J].南京师大学报(自然科学版),2012,35(01):84-88.
 Peng Wanxin,Sun Yaoxiang,Chen Chen,et al.Construction of Recombinant pDsRed-LC3 Fusion Protein and Its Expression and Localization in HepG2 Cell[J].Journal of Nanjing Normal University(Natural Science Edition),2012,35(01):84-88.
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pDsRED-LC3真核表达载体的构建及其在肝癌细胞系HepG2中的表达和定位()
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《南京师大学报(自然科学版)》[ISSN:1001-4616/CN:32-1239/N]

卷:
第35卷
期数:
2012年01期
页码:
84-88
栏目:
生命科学
出版日期:
2012-03-20

文章信息/Info

Title:
Construction of Recombinant pDsRed-LC3 Fusion Protein and Its Expression and Localization in HepG2 Cell
作者:
彭琬昕;孙瑶湘;陈琛;金洁;邵根宝;王嫘;
江苏大学基础医学与医学技术学院,江苏镇江212013
Author(s):
Peng WanxinSun YaoxiangChen ChenJin JieShao GenbaoWang Lei
School of Medical Sciences and Laboratory Medicine,Jiangsu University,Zhenjiang 212013,China
关键词:
红色荧光蛋白MAP-LC3自噬肝癌
Keywords:
red fluorescent proteinMAP-LC3autophagyhepatocarcinoma
分类号:
R735.7
摘要:
本实验根据已知的人MAP-LC3 序列,以人脑胶质瘤U251 细胞cDNA 为模板扩增出去除终止密码子的 MAP-LC3 片段,定向克隆至红色荧光融合蛋白载体pDsRed-N1 质粒中,构建了重组质粒pDsRed-LC3. 将pDsRed- LC3 转染HepG2 细胞, 48 h 后用Western Blot、激光共聚焦显微镜检测融合蛋白在细胞内的表达和分布情况,并观察血清饥饿时细胞发生自噬的情况. 结果表明: ( 1) 成功构建pDsRFP-LC3 真核表达载体,该载体能在人肝癌细胞中表达; ( 2) 血清饥饿应激实验证明,该载体可以良好地指示自噬泡的形成过程,为研究肿瘤细胞的自噬发生情况提供了一个重要而方便的工具.
Abstract:
In this study,according to the nucleotide sequence of MAP-LC3,we successfully constructed the vector that expressed the fusion protein of MAP-LC3 and red fluorescent protein ( RFP) in mammalian cells. The recombinant plasmid was identified by restriction endonuclease enzyme analysis and DNA sequence analysis. The plasmid was transfected into HepG2 cells,and the expression and distribution of the MAP-LC3 were observed with Confocal Laser Scanning Microscopy ( CLSM) after 48 hours. All of the results indicated that this vector was highly expressed in the HepG2 cells. The red fluorescence was found over the cytoplasm by CLSM. Therefore,we had a conclusion that a recombinant plasmid of MAP-LC3 and RFP was constructed successfully,and it would be an important and convenient tool to study the process of autophagy in HCC.

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备注/Memo

备注/Memo:
基金项目:国家自然基金( 31100964) 、江苏省高校科学研究基金( 09KJB310002) 、江苏大学高级人才科研启动基金( 10JDG045) .
通讯联系人:彭琬昕,博士,讲师,研究方向: 肿瘤细胞生物学. E-mail: pengwanxin@ ujs. edu. cn
更新日期/Last Update: 2013-03-11