中文摘要：

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,western 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基因已完全正确亚克隆到pEGFP—N3;LC3-EGFP融合蛋白能够在HEK293T细胞中表达,并主要定位于细胞质中。结论成功构建具有报告基因—增强绿色荧光蛋白基因的重组真核表达载体pLC3-EGFP并在HEK293T细胞中很好地表达,实验结果为研究细胞自噬的进程及机制提供了实验基础。

英文摘要：

[ 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 observed under fluorescent microscope, and the expression LC3-EGFP protein was detected by western blotting. [Results] The PCR amplification yielded a single band of about 378 base pairs, sequence analysis of the PCR products revealed that it was consistent with that of the references published [GenBank （NM_022818）]. Further- more, a recombinant plasmid pLC3-EGFP for eukaryotic expression was also successfully constructed after subcloning. LC3-EGFP fusion protein was expressed in the HEK293T cells and mainly presented in the cytoplasm after transfec- tion with pLC3-EGFP. [Conclusion] The recombinant eukaryotic expression vector of pLC3-EGFP has been constructed successfully and EGFP tagged LC3 is immediately expressed in the HEK293T cells and localized in the cytoplasm, which lay the foundation for further studying the biological progression and mechanisms of cell autophagy.