哺乳动物雷帕霉素靶蛋白重组RNAi慢病毒载体的构建

Build of lentiviral mediated RNA interference of mammalian target of rapamycin

目的构建哺乳动物雷帕霉素靶蛋白(mTOR)重组RNA干扰(RNAi)慢病毒表达载体。方法按照RNAi设计规则,针对mTOR基因设计4个干扰靶点和阴性对照序列(FAM),先用人工合成寡核甘酸片段,通过PCR拼接的方法,即可获得小干扰RNA(siRNA)有效片段,采用Lipofectamine 2000转染试剂,对肺腺癌A549细胞进行转染,1d后,通过高倍荧光显微镜观察增强型绿色荧光蛋白在肺腺癌A549细胞中的表达情况。采用半定量RT-PCR方法,检测mTOR基因在1d后mRNA水平的表达,采用Western blot检测2d后蛋白表达水平,从而筛选出最高效的干扰靶序列,将其合成双链DNA,通过pGCL-GFP载体,与pHelper 1.0和pHelper 2.0质粒共同组成载体系统,进一步转染293T细胞,最终包装后产生慢病毒,通过Western blot方法检测GFP蛋白表达水平来检测293T细胞中的病毒滴度,并进行活性鉴定。结果 mTOR基因的高效siRNA干扰靶点被成功筛选出;mTOR siRNA感染体外培养的人肺腺癌A549细胞后,不管是从mRNA水平,还是蛋白水平,该基因都明显沉默;该基因mTOR siRNA的慢病毒载体被成功构建,同时收获病毒上清,并检测出病毒滴度为1×108 UT/mL。结论 mTOR siRNA感染体外培养的人肺腺癌A549细胞,能够导致mTOR基因明显沉默;该基因mTOR siRNA的慢病毒载体被成功构建。

Objective To build of lentiviral mediated RNA interference of mammalian target of rapamcin （mTOR） ,because the mammalian target of rapamycin plays a important role in tumor development and the signal path .Methods According to RNA in-terference （RNAi） design rules Completely ,in view of the gene called mTOR was desiged four interference targets and Negative control （FAM ） sequence ,first of all ,synthetic oligonucleotides nucleotide fragments with artificial ,and can obtain siRNA fragments effectively by the method of PCR joining together ,then undertake transfection on lung adenocarcinoma A 549 cell by Lipofectamine transfection reagent 2000 .To begin to observe the enhanced green fluorescent protein expression in lung adenocarcinoma A 549 cells by fluorescence microscopy at high magnification after 1 days .We can use semi-quantitative RT-PCR method ,and detect of mTOR gene expression of mRNA level after 1 days ,meanwhile ,testing the expression of protein levels by Western Blot after 2 days ,in or-der to select the most efficient interference target sequence ,,afterwards ,synthetic double-stranded DNA ,and it can be make up vec-tor system with plasmid pHelper 1 .0 and pHelper 2 .0 by the pGCL-GFP carrier ,further transfect 293 T cells ,at last produce lenti-viral after packaging ,then detection GFP protein expression levels by Western-Blot method ,and consequently detect the virus drops degree of 293T cells ,at the same time ,identify the activity .Results The high efficiently target of mTOR gene has been successfully selected ;mTOR siRNA infecte of human lung adenocarcinoma A549 cells in vitro culture ,and the gene is obviously silence no mat-ter from the mRNA level or protein level ;3 mTOR gene lentivirus siRNA carrier was successfully build .geting virus supernatant al-so ,and virus drops to 1 ＆#215; 108 UT/mL .Conclusion MTOR siRNA infected of human lung adenocarcinoma A549 cells in vitro cul-ture ,and could lead to mTOR gene obviously silence ;The construction was successfully gene mTOR siRNA lentivirus vectors .