增殖抑制基因（HSG）RNAi 慢病毒载体的构建与鉴定

摘要

Objective To construct and identify the efficacy of a lentiviral vector harboring RNA interference sequence targeting hyperplasia suppressor gene(HSG). Methods Four siRNA sequences targeting HSG mRNA were designed. The lentivirus vectors of GCSIL-GFP-HSG were constructed and confirmed by DNA sequencing. The 293T cells were co-transfected with pGCSIL-GFP-HSG,pHelper1. 0 and pHelper2. 0 in order to produce virus stocks. The titer of the virus was also tested. The lentivirus was transfected to human A549 lung adenocarcinoma cells. The expression of HSG gene was analyzed by real-time PCR. Results DNA sequencing suggested that the DNA sequences were consisted with the design,which meant that the RNAi sequence targeting the human HSG gene was correct. Examination of the co-transfected cells by fluorescence microscopy suggested that the cells grew well and had strong fluorescence intensity. The titer of the virus was 3× 108 TU / ml. Real-time PCR showed that the expression of the HSG gene was knockdown after the lentivirus transfected to A549 cells. Conclusions The lentiviral vector of the HSG gene of Homo sapiens was successfully constructed,which could be further used in oncology.%目的：为研究 HSG 基因不同表达程度对肺癌细胞的影响，构建并鉴定 HSG 基因 RNA 干扰慢病毒表达载体，以建立 HSG 基因沉默的人肺癌细胞株。方法针对 HSG mRNA 设计了4条 siRNA，并构建 pGCSIL-GFP-HSG 慢病毒质粒，通过测序鉴定。采用构建的 pGCSIL-GFP-HSG，pHelper1.0和pHelper2.0共感染293T 细胞，包装产生慢病毒，测定其滴度。将慢病毒转染人肺腺癌细胞株 A549，通过 real-time PCR 分析 HSG 基因表达。结果测序结果显示，DNA 序列与实验要求序列一致，提示插入人HSG 基因 RNAi 序列正确。荧光显微镜观察转染慢病毒包装质粒后的细胞，见细胞生长良好，荧光强度强烈。测定病毒滴度为3×108 TU / ml。real-time PCR 分析提示 RNA 干扰病毒感染 A549细胞株后，HSG基因表达显著下调。结论人 HSG 基因 RNAi 慢病毒载体构建成功，可用于肿瘤学的进一步应用。