自噬相关基因LC3B真核表达载体构建及胃癌细胞自噬的观察

Construction and Identification of Eukaryotic Expression Vector of Human Autophagy-related LC3B

目的:构建人自噬相关基因微管相关蛋白1轻链3B(microtubule associated protein 1 light chain 3B,LC3B)真核表达载体,并鉴定其生物学功能。方法:通过RT-PCR方法扩增得到人LC3B c DNA,应用基因重组技术构建p EGFP-C1-LC3B真核表达载体,通过酶切和测序进行鉴定。将p EGFP-N1-LC3B表达载体转染胃癌细胞MKN1,倒置荧光显微镜观察饥饿诱导后细胞中EGFP-LC3B的表达及分布变化。结果:通过测序鉴定,p EGFP-N1-LC3B真核表达载体序列正确,编码框正确。转染后的MKN1细胞经饥饿诱导后,检测发现EGFP-LC3B由散在分布向点状分布改变,即生成细胞自噬体。结论:成功构建了人自噬相关基因LC3B真核表达载体,并证实饥饿诱导胃癌细胞自噬的发生,为进一步研究自噬在肿瘤中的作用机制奠定了基础。

Objective： To construct the eukaryotic expression vector of human autophagy-related micortubule associated protein 1 light chain 3B （LC3B） gene and to detect its expression in gastric cancer cell line MKN1. Methods： LC3B cDNA was amplified by RT-PCR. The eukaryotic expression vector of pEGFP-N1-LC3B was constructed by gene recombination technique and the recombinant plasmid was verified by restriction enzyme analysis and sequencing. The plasmid was transfected into MKN1 cells and the expression of EGFP-LC3B were detected by fluorescence microscopy. Results： The sequences and open read frames of the vector were completely in accordance with experimental design. After starvation induction, the distribution of EGFP-LC3B from diffused to punctuate were observed. Conclusions： Eukaryotic expression vector of human LC3B gene is successfully constructed. It provides an experimental base for further research.