人自噬相关基因LC3B真核表达载体的构建及鉴定

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

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。

Objective：To construct the eukaryotie expression vector with human autophagy-related microtubule-associated protein 1 light chain 3B （ LC3 B） 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-LC3 B 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.