期刊论文

中文

中国现代医学杂志

1005-8982

2011

21

36

4483-4486,4492

10.3969/j.issn.1005-8982.2011.36.001

(81172712):国家自然科学基金 (11JJ6078):湖南省自然科学基金 (09B087):湖南省教育厅优秀青年基金

本校为第一机构

目的 构建人微管相关蛋白轻链3(LC3)与增强型绿色荧光蛋白(EGFP)融合表达的真核表达载体(pLC3-EGFP)并在哺乳动物细胞中表达,为研究自噬的过程及机制提供实验基础.方法 应用RT-PCR从人胚肾细胞系HEK293T细胞中扩增全长LC3基因,将产物LC3 cDNA片段亚克隆入载体pEGFP-N3,筛选阳性克隆,提取质粒测序进行鉴定.将表达质粒pLC3 -EGFP转染HEK293T细胞,荧光显微镜观察融合蛋白在细胞内的表达与定位,westem blot检测LC3-EGFP蛋白的表达.结果 测序结果表明,从人胚肾HEK293T细胞中所获得的LC3 cDNAs含有378个碱基,与Genbank( NM 022818)序列完全一致.构建的重组真核表达载体pLC3-EGFP经鉴定证实LC3基因已完...

[ Objective ] To construct eukaryotic expression vector pLC3-EGFP and to observe the expression in human embryonic kidney cell line HEK293T cells. [Methods ] The full length gene of human microtubule-associat- ed protein 1 hght chain3 （LC3） was amplified from HEK293T by RT-PCR. Then the cDNA fragment was subcloned into pEGFP-N3 vector and the positive clones were selected and sequenced. The recombinant plasmid pLC3-EGFP was transfected into the HEK293T cells using VigoFect transfection agent. Localization of the fusion protein LC3- EGFP was o...