四环素诱导调控的腺病毒载体系统的构建及其应用

目的构建四环素诱导调控基因表达的腺病毒载体系统,评估该载体系统的高效性。方法利用PCR、限制性内切酶酶切、连接等分子生物学手段构建四环素诱导调控体系中调节因子序列、应答元件序列的穿梭载体。通过同源重组的方法将载体上的四环素调控因子以及应答元件序列整合于腺病毒基因组。利用RT-PCR检测病毒载体上四环素调节因子（rtTA和tTA）在Hela细胞中的转录水平;荧光素酶活性测定分析腺病毒载体系统的效率。在不同浓度强力霉素的诱导下,检测荧光素酶报告基因活性变化,分析可诱导调控腺病毒系统的有效性和敏感性。结果调控性腺病毒AdrtTA^Tet-on与Ad-tTA^Tet-off分别感染Hela细胞,逆转录PCR检测鉴定腺病毒载体Ad-rtTA^Tet-on与Ad-tTA^Tet-off的表达克隆用于调控应答性腺病毒Ad-TREmod/minCMV△-LUC、Ad-TREmod/U6-shLUC。在强力霉素的诱导下调控性腺病毒Ad-rtTA^Tet-on与Ad-tTA^Tet-on能够开启或关闭应答性腺病毒Ad-TREmod/minCMinCMV△-LUC、Ad-TREmod/U6-shLUC的表达,从而达到调控荧光素酶的表达或RNA干扰荧光素酶基因的目的。结论构建可诱导调控的腺病毒载体系统,该系统能够在强力霉素的调控下开/关目的基因、特异性干扰目的基因shRNA片段的表达。

Construction and application of adenovirus-based tetracycline-inducible expression system

Objective To construct adenovirus-based tetracycline-inducible system for both specific gene expression,and to evaluate the efficiency of the system.Methods The sequences of the tetracycline regulatory factors and the response elements were amplified,enzymatic digested and integrated separately into the recombinant shuttle vector.Through homologous recombination,the tetracycline regulatory factors and the response elements were integrated into adenoviral DNA.RT-PCR was adopted to detect transcription level of the tetracycline regulatory factors rtTA and tTA gene in Hela cells.The luciferase expression level was examined by lueiferase activity assayin Hcla cells to evaluate the Ad-TREmod/minCMVA-LUC and Ad-TREmod/U6-shLUC.By the induction of gradient concentration of doxycycline,luciferase activity was examined to analyze the effectiveness and sensitiveness of the adenovirus-based tetracycline-inducible system.Results The regulatory adenovirus Ad-rtTA^Tet-on and Ad-tTA^Tet-off were used to regulate responsive adenovirus Ad-TREmod/minCMVA-LUC,Ad-TREmod/U6-shLUC by RT-PCR analysis in adenovirus-infected Hela cells.Under the induction of doxycycline,regulatory adenovirus(Adrt TA^Tet-on,Ad-tTA^Tet-°ff) could turn on/off responsive adenovirus（Ad-TREmod/minCMV△-LUC,Ad-TREmod/U6-shLUC）,and achieve the goal of overcxpression or RNA interference of luciferase gene.Conclusion The successfully constructed system of adenovirus-based tetracycline-inducible expression can control turning on/off of tetracycline derivative（doxycycline） expression or the knockdown of target gene.