VASP基因RNA干扰慢病毒表达载体的构建与鉴定  

Construction of lentivirus-mediated RNAi vector targeting vasodilator-stimulated phosphoprotein

作　　者：王静[1] 魏蕾[2] 邱堃[1] WANG Jing;WEI Lei;QIU Kun(Department of Pathology,Wuhan No.1 Hospital,Hubei Province,Wuhan 430022,China;Department of Pathology and Pathophysiology,School of Basic Medical Sciences,Wuhan University,Hubei Province,Wuhan 430071,China)

机构地区：[1]武汉市第一医院病理科,湖北武汉430022 [2]武汉大学基础医学院病理学与病理生理学系,湖北武汉430071

出　　处：《中国医药导报》2018年第25期9-12,共4页China Medical Herald

基　　金：湖北省自然科学基金项目(2014CFB340)

摘　　要：目的构建靶向血管扩张刺激磷蛋白(VASP)基因RNA干扰慢病毒表达载体。方法将前期构建的pcDNA6.2-miRVASP进行测序鉴定,筛选阳性克隆。通过Gateway技术中的BP反应及LR反应,将携带miRVASP的表达框克隆至慢病毒目的载体pLenti6/V5-DEST,构建靶向VASP基因的RNA干扰慢病毒表达载体pLenti6/V5-miRVASP;接着将其与慢病毒包装混合物共转染293FT细胞,收集病毒颗粒,侵染胃癌BGC-823细胞。结果慢病毒表达载体测序结果表明,构建的载体与预期完全一致。用收集的携带miRVASP表达框的慢病毒颗粒侵染BGC-823细胞,荧光显微镜下可见多量细胞表达强绿色荧光,证明包装产生的慢病毒颗粒能侵染BGC-823细胞。结论成功构建了靶向VASP基因的RNA干扰慢病毒表达载体,并包装产生慢病毒颗粒原液,成功侵染BGC-823细胞,为进一步在体内实验中研究VASP在胃癌侵袭转移中的作用奠定了基础。Objective To construct the lentiviral RNA interference expression vector targeting vasodilator-stimulated phosphoprotein（VASP） gene. Methods The pre-constructed pcDNA6.2-miRVASP was identified by DNA sequencing and the positive clones were screened. The miRVASP fragment was cloned into destination vector pLenti6/V5-DEST by Gateway techology, including BP and LR reaction, RNA interference lentiviral vector pLenti6/V5-miRVASP targeting VASP gene was constructed. Then the 293 FT cells were co-transfected with the plasmid lentiviral expression vector pLenti6/V5-miRVASP and lentiviral packaging mix. The virus particles were collected and infected into gastric cancer BGC-823 cells. Results The constructed lentiviral expression vector pLenti6/V5-miRVASP was introduced into E.coli Stbl3 for amplification. DNA sequencing results showed that the constructed vector was exactly as expected. BGC-823 cells were infected with the collected lentiviral stocks carrying the miRVASP expression cassette. The results showed that a large number of cells could express strong green fluorescence, demonstrated that the packaged lentiviral stocks could infect BGC-823 cells. Conclusion The RNA interference lentiviral vector targeting VASP gene is successfully constructed and packaged as the lentiviral stocks to successfully infect BGC-823 cells, which lay the foundation for the further study of the role of VASP in gastric cancer metastasis in vivo.

关 键 词：血管扩张刺激磷蛋白 RNA干扰 慢病毒载体 

分 类 号：R737[医药卫生—肿瘤]