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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 replication-defective adenovirus Ad-RUDsRed with RU486-regulated red fluorescent protein (DsRed) for tumor gene therapy. Methods: Adenovirus shuttle plasmid pDC-RUDsRed with RU486 regulatory system and DsRed gene was constructed. The positive clones were screened and digested with restriction endonucleases and sequenced. The adenovirus Ad-RUDsRed was amplified by AdMaxTM system, and then amplified, purified and titrated. Ad-RUDsRed adenovirus Ad-RUDsRed cells were infected with Ad6 + 10-8, 1 × 10-7 and 1 × 10-6mol / L RU486 induction, the use of fluorescence microscopy and flow cytometry detection of Ad-RUDsRed regulatory function. Results: The results of PCR confirmed that adenovirus vector Ad-RUDsRed was successfully constructed with the titer of 3.46 × 1010pfu / mL. DsRed expression was correlated with RU486 dose, with no RU486 reporter DsRed almost no expression. Given the inducer RU486, the adenovirus vector can induce the expression of red fluorescent protein. When RU486 dose reached 1 × 10-6mol / L, the expression of DsRed reached the maximum. CONCLUSION: Ad-RUDsRed, a replication-defective adenovirus carrying RU486-regulated DsRed, was successfully constructed and the expression of DsRed was induced by RU486, which laid the foundation for the gene therapy of tumors.