王静 魏蕾 邱堃

[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。

[关键词] 血管扩张刺激磷蛋白；RNA干扰；慢病毒载体

[中图分类号] R737 [文献标识码] A [文章编号] 1673-7210（2018）09（a）-0009-04

[Abstract] 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 293FT 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.

[Key words] VASP； RNAi； Lentiviral vector

胃癌是最常见的消化道恶性肿瘤之一。在我国，胃癌发病率居各类恶性肿瘤第二位[1]。尽管近年来胃癌病理遗传学和分子机制研究取得了许多进展，但胃癌转移仍然是癌症相关死亡的主要原因之一。肿瘤转移是一个多步骤的过程[2]，以间充质模式迁移的肿瘤细胞，首先形成由肌动蛋白丝（F-actin）网络组装驱动的细胞膜突起（伪足）；接着在原癌基因如c-Src及c-Abl激酶等分子和细胞骨架调节蛋白协调作用下，促进细胞迁移和侵袭[3]。血管扩张刺激磷蛋白（vasodilator-stimulated phosphoprotein，VASP）是Ena/VASP蛋白家族成员之一[4]。其EVH2结构域介导其与球状肌动蛋白（G-actin）及F-actin的结合。……