可调控腺病毒载体Ad-RUDsRed构建及其在结肠癌SW620细胞中表达的实验研究

Construction of RU486 inducible recombinant adenovirus Ad-RUDsRed and identification of expression of SW620 cells in vitro

目的:构建带有RU486可调控红色荧光蛋白(DsRed)的复制缺陷型腺病毒Ad-RUDsRed,用于肿瘤基因治疗。方法:构建带有RU486调控系统和DsRed基因的腺病毒穿梭质粒pDC-RUDsRed,筛选阳性克隆,酶切、测序鉴定。利用AdMaxTM系统重组得到腺病毒Ad-RUDsRed,进行扩增、纯化和滴度测定;腺病毒Ad-RUDsRed感染SW620细胞细胞株后分别加入0、1×10-10、1×10-9、1×10-8、1×10-7和1×10-6mol/L的RU486诱导,利用荧光显微镜和流式细胞仪检测Ad-RUDsRed调控功能。结果:PCR结果证实,成功构建可调控腺病毒载体Ad-RUDsRed,病毒滴度3.46×1010pfu/mL。DsRed的表达与RU486剂量相关,无RU486时,报告基因DsRed几乎没有表达。给予诱导剂RU486,腺病毒载体可以诱导表达红色荧光蛋白,RU486剂量达1×10-6mol/L时DsRed的表达量达到最大值。结论:成功构建携带RU486可调控DsRed的复制缺陷型腺病毒Ad-RUDsRed,通过RU486可以诱导调控DsRed的表达,为肿瘤的基因治疗奠定了基础。

OBJECTIVE：To construct of a RU486 inducible recombinant adenovirus Ad-RUDsRed and identificate the expression in vitro for future tumor gene therapy.METHODS：The adenovirus shuttle plasmid pDC-RUDsRed carrying RU486-inducible system was constructed by molecular biology methods.The positive clone was identified by restriction endonuclease digestion and further confirmed by sequencing.The recombinant adenovirus was produced by a AdMaxTM system and purified by CsCl density gradient centrifugation and its titer was determined by end point dilution assay.The regulating function of recombinant adenovirus vector was checked at different dosages of RU486 （0,1×10^-10,1×10^-9,1×10^-8,1×10^-7 or 1×10^-6 mol/L） in infected SW620cells by fluorescence microscopy and FCM.RESULTS：The vector Ad-RUDsRed was confirmed by PCR.The virus titer of Ad-RUDsRed was 3.46×10^10 pfu/mL.RU486 induced Ad-RUDsRed expression in a dose-dependent manner.Without RU486,no significant DsRed protein activation was observed,whereas in the presence of RU486,the activation of the red fluorescent protein was visible.When the dose of RU486 arrived to 1×10-6 mol/L,the expression of DsRed increased to maximum.CONCLUSIONS：The replication-defective recombinant adenovirus vector encoding DsRed regulated by RU486 （Ad-RUDsRed） is constructed successfully.The virus vector provides a useful tool for tumor gene therapy in vivo.