人自噬相关基因LC3B真核表达载体的构建及鉴定

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,MAP1LC3B)真核表达栽体,并鉴定其生物学功能。方法:通过RT-PCR方法扩增得到人MAP1LC3B cDNA;应用基因重组技术构建pEGFP-N1-LC3B真核表达载体,通过酶切和测序进行鉴定;倒置荧光显微镜下观察EBBS诱导4 h后pEGFP-N1-LC3B表达载体转染的HepG2.2.15细胞质中GFP-LC3B分布的变化。结果:通过测序鉴定,pEGFP-N1-LC3B真核表达载体序列正确,编码框正确;转染后的HepG2.2.15细胞经EBBS诱导4 h后,倒置荧光显微镜检测发现GFP-LC3B由散在分布向点状分布改变。结论:成功构建了人自噬相关基因LC3B真核表达载体,为进一步研究自噬在乙型肝炎病毒中的作用机制奠定了基础。

Construction and identification of eukaryotic expression vector recombined with human autophagy-related LC3B gene

Objective:To construct the eukaryotie expression vector with human autophagy-related microtubule-associated protein 1 light chain 3B(LC3B) gene and to detect its expression in HepG2.2.15 cells. 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 positive clones were transfected into HepG2.2.15 cells,and the distribution of GFP-LC3B in HepG2.2.15 cells were observed under fluorescence microscopy after EBBS inducing 4 hours. Results:The sequences and open read frames of the vector were completely in accordance with experimental design.After inducting 4 hours,the change-from diffused to punctuate distribution of GFP-LC3B in HepG2.2.15 cells were observed under fluorescence microscopy. Conclusions:Eukaryotic expression vector of human LC3B gene is successfully constructed.It provides an experimental base for further research work.