带有荧光素酶报告基因的可调控重组腺病毒载体的构建及表达

Construction of RU486 regulatable adenoviral vector containing luciferase gene and its expression in vitro

目的:构建带有萤火虫荧光素酶报告基因的可调控腺病毒载体,并在SW620结肠癌细胞株中观察其调控表达.方法:将萤火虫荧光素酶luc基因和启动子,以及RU486调控系统构建成单一的穿梭载体PDC-RULUC,将其与Admax腺病毒包装系统的辅助质粒共转染293细胞后,构建出可调控的重组腺病毒载体.扩增后用最终稀释法测定了其滴度,并在SW620细胞中检测了其基因调控效果.结果:成功地构建了携带有RU486调控系统的可调控腺病毒载体Ad-RULUC,病毒滴度达到5.2×1013pfu/L.当给予诱导剂RU486后,腺病毒载体可以诱导表达荧光素酶,并在一定范围内两者呈正比,而没有RU486时,几乎没有报告基因的表达.结论:腺病毒Ad-RULUC能调控外源基因的表达,为基因调控研究和基因治疗提供了良好的工具.

AIM： To construct RU486 regulatable replicationdefective recombinant adenovirus containing luciferase gene, and to detect its expression in SW620 cells. METHODS： The single shuttle vector named PDC-RULUC was constructed with molecular biological methods, which contained firefly luciferase reporter gene, promoter and RU486-inducible system. Recombinant adenovirus was produced after cotransfection of the shutter vector PDCRULUC and adenovirus DNA helper plasmid pBHGlox△E1,3Cre into 293 cells. After the amplification, the recombinant adenoviral titer was determined by end point dilution assay. Expression in SW620 cells was tested by Luciferase Reporter Assay. RESULTS： The regulatable recombinant adenovirus vector containing luciferase gene was identified by PCR. The virus titer of Ad-RULUC was 5.2 × 10^13 pfu/L in the absence of RU486, no significant luciferase activation was observed, whereas in the presence of RU486,activation of the luciferase was positively correlated with the inducer under definite range. CONCLUSION： A RU486 regulatable recombinant adenoviral vector containing luciferase gene is successfully constructed, which provides a good basis for further research on gene regulation and gene therapy.