逆转录病毒Pmscv/Hyg介导的RNA干扰表达载体的构建与鉴定

目的构建逆转录病毒Pmscv/Hyg介导的RNA干扰表达载体,并在HEK293细胞株中观察其基因沉默效果。方法PCR方法扩增人U6启动子,下游引入核酸内切酶位点BamHI、SalI,反向插入质粒Pmscv/Hyg的3′端LTR上游。扩增EGFP基因,插入载体Pmscv/Hyg的多克隆位点。合成干扰P53基因表达的寡核苷酸序列,退火复性后插入U6启动子下游BamHI、SalI酶切位点间。重组质粒经酶切、测序鉴定正确后转染病毒包装细胞PT67,产生具有一次感染能力的病毒颗粒。病毒上清感染靶细胞HEK293,经潮霉素筛选,RT-PCR和western blot检测靶基因P53表达情况。结果质粒酶切及DNA测序表明成功构建重组病毒载体,并在PT67细胞中包装成具有一次感染能力的逆转录病毒。经Hygromycin筛选,HEK293细胞中P53蛋白和mRNA表达显著下调。结论成功构建以Pmscv/Hyg为基础的RNA干扰表达载体,重组载体包装出的逆转录病毒可有效介导基因沉默。

Construction and Identification of a Novel Retroviral Vector Delivery shRNA based on the Pmscv/Hyg

Objectives To construct a novel retroviral vector delivery shRNA based on the Pmscv/Hyg vector,and investigate its effect of gene silencing on HEK293 cells. Methods Human U6 promoter containing the introduced BamHI and SalI sites was amplified from genomic DNA and reversely inserted into the upstream of the 3＇LTR in Pmscv/Hyg. The EGFP gene was cloned from plasmid PEGFP-N1 and inserted into the multiclone sites in Pmscv/Hyg. The oligonucleoti- des encoding the human P53 siRNA was annealed and ligated into the BamHI and SalI sites. After screening and amplification, the recombinant retroviral plasmid was obtained and transfected into PT67 cells. The HEK293 cells were transduced with retrovirus supernatant. The positive clones were selected by Hygromycin. The expression of P53 mRNA and protein were evaluated by RT-PCR and Western blotting respectively. Results The novel recombinant vector was confirmed by agarose gel electrophoresis after restriction enzyme digestion and by sequence analysis. RTPCR and Western blot analysis demonstrated a remarkable downregulation of P53 mRNA and protein in HEK293 cells infected with Pmscv/P53shRNA. Conclusion The retroviral vector Pmscv/EGFP/shRNA has been successfully Constructed, and he retroviral system is capable of delivering short interference RNA against target gene.