自噬标志分子MAP1LC3的重组载体构建及表达分析

Establishment of the LC3 Recombinant Vector and Its Expression in Prokaryotic and Eukaryotic Cells

目的:为深入研究细胞自噬的发生过程及机制,克隆人微管相关蛋白1轻链3-β(microtubule associated protein 1 lightchain 3,MAP1LC3,简称LC3)基因,并构建原核和真核重组质粒用于后续的研究。方法:首先RT-PCR方法从人食管癌9706细胞基因组中克隆LC3基因,并分别连接到pET-32a载体和pEGFP-N3载体上,形成重组质粒。前者用IPTG(终浓度1mmol/L)诱导表达,后者进行细胞转染实验。结果:IPTG诱导原核重组质粒在E.coli内实现表达,pEGFP-N3-LC3转染到A549人肺癌细胞36h和鲤鱼上皮瘤细胞系(Epithelioma papulosum cyprinid,EPC)后,均检测到明显的绿色荧光信号,且在细胞质和胞核内均有分布。结论:成功构建了人源LC3重组表达载体,为研究高等和低等脊椎动物细胞自噬机制及机理过程提供了很好的研究工具。

Objective：Microtubule associated protein 1 light chain 3 （MAPI LC3, for short LC3 ）is the first identified autophagosome mem- brane protein. In this paper,the human LC3 gene was cloned and the Prokaryotic and Eukaryotic recombinant vectors were constructed forfurther research. Method：The human LC3 gene was cloned from esophagus cancer cell 9706 with RT - PCR and then the pET -32a - LC3 and pEGFP - N3 - LC3 recombinant plasmid vectors were constructed. The pET32a - LC3 plasmid was induced by IPTG（ 1 mmol/L） in E- ~ coli cells ,while the pEGFP - N3 - LC3 plasmid DNA was transiently transfected into the A^9 lung cancer cells and one fish cell line（ Ep- ithelioma papulosum cyprinid, EPC ） cells, respectively. Result： The LC3 protein was expressed in pET32a - LC3 transformed E. coli cells. Fluorescent microscope examination showed that fluorescent signals could be detected in both the cytoplasm and nucleus of tranfected cells after 36 hours. Conclusion： The LC3 recombinant expression vectors were established, which would provide a good cell research model for future vertebrate cell autophagy mechanisms and biological processes.