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

Construction of Recombinant pDsRed-LC3 Fusion Protein and Its Expression and Localization in HepG2 Cell

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.

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.