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小鼠硒蛋白mSelK基因敲减shRNA慢病毒重组载体的构建及鉴定

发表时间:2018-02-11  浏览量:932  下载量:100
全部作者: 张会苓,孟雪莲,贲松彬,王广超,郇丰宁,冯琳琳,陈长兰
作者单位: 辽宁大学生命科学院;辽宁大学药学院
摘 要: 目的:构建小鼠硒蛋白mSelK 基因敲减shRNA 慢病毒重组载体,以研究硒蛋白mSelK 基因敲减对小鼠细胞株的影响,对研究硒蛋白mSelK 的分子功能,尤其是在巨噬细胞等免疫细胞中的分子功能具有重要意义。方法:将初始载体pLKO.1-TRC 中Age I 和EcoR I 酶切位点之间1.9 kb 的插入片段切除,再插入mSelK的shRNA 退火片段构建pLKO.1-mSelK-shRNA 载体。将pLKO.1-mSelK-shRNA 载体与包装质粒psPAX2、包膜质粒pMD2.G 共同转染HEK-293T 细胞,从而构建mSelK 基因shRNA 表达敲减慢病毒重组载体pLKO.1-mSelK-shRNA. 用逆转录RCR(reverse transcription-PCR,RT-PCR)和Western blotting 法考察mSelK表达敲减慢病毒载体对RAW264.7 细胞中mSelK 的敲减能力。结果:在单核细胞RAW264.7 中mSelK 的mRNA和蛋白含量显著降低,可以看出,慢病毒基因敲减mSelK 的效果非常明显。结论:采用pLKO.1 慢病毒载体表达系统成功构建了高效敲减小鼠硒蛋白mSelK 的慢病毒重组载体pLKO.1-mSelK-shRNA,为进一步研究小鼠硒蛋白mSelK 的分子功能奠定了实验基础。
关 键 词: 分子生物学;小鼠硒蛋白;mSelK;shRNA;慢病毒载体
Title: Construction and identification of the lentivirus recombinant vector of mouse selenoprotein mSelK gene knockdown shRNA
Author: ZHANG Huiling, MENG Xuelian, BEN Songbin, WANG Guangchao, HUAN Fengning, FENG Linlin, CHEN Changlan
Organization: School of Life Science, Liaoning University; School of Pharmacy, Liaoning University
Abstract: Objective: The construction of the lentivirus recombinant vector of mouse selenoprotein mSelK gene knockdown shRNA is important for the researches on the molecular functions of mSelK, especially in macrophage and other immune cells, and the studies of its influence on mouse cell lines. Methods: The 1.9 kb insertion fragment of the original pLKO.1-TRC cloning vector was excised by digestion with Age I and EcoR I, and the annealing shRNA oligos of mSelK were cloned into the Age I and EcoR I sites to construct pLKO.1-mSelK-shRNA vector. Then the constructed pLKO.1-mSelK-shRNA vector, psPAX2 packaging plasmid and pMD2.G enveloping plasmid were co-transfected into HEK-293T cells to construct the mSelK gene shRNA lentiviral recombinant vector pLKO.1-mSelK-shRNA. The lentivirus recombinant vector’s mRNA knockdown effect of mSelK in monocyte cell strain RAW264.7 was detected by reverse transcription-RCR (RT-PCR) and Western blotting. Results: The expressions of mRNA and protein in mSelK in RAW264.7 cells are significantly reduced than that in the control group, and it means that the knockdown effect of the lentivirus recombinant vector on mSelK is rather effectively. Conclusion: The lentivirus recombinant vector pLKO.1-mSelK-shRNA, which could knockdown mice selenoprotein mSelK, is successfully constructed using pLKO.1 lentivirus recombinant vector system. And the construction of the recombinant vector makes a foundation for the further study of the molecular function of mice selenoprotein mSelK.
Key words: molecular biology; mouse selenoprotein; mSelK; shRNA; lentivirus vector
发表期数: 2018年2月第3期
引用格式: 张会苓,孟雪莲,贲松彬,等. 小鼠硒蛋白mSelK基因敲减shRNA慢病毒重组载体的构建及鉴定[J]. 中国科技论文在线精品论文,2018,11(3):265-270.
 
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