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目的构建携带绿色荧光蛋白(GFP)标签的表达PLCε基因shRNA的重组腺病毒质粒Ad-U6-PLCε,为应用基因沉默技术研究膀胱癌的基因治疗奠定基础。方法将干扰质粒pGenesil-PLCε及阴性对照质粒pGenesil-NP的表达启动子U6及shRNA序列克隆至带有增强型绿色荧光蛋白(EGFP)报告基因的穿梭质粒pAdTrack,经酶切及测序鉴定正确后,将重组穿梭质粒经PmeI线性化,转化感受态AdEasier。重组pAdEasy-U6-PLCε质粒经PacI线性化后,转染HEK-293细胞,包装重组腺病毒Ad-U6-PLCε,经大量扩增后测定病毒滴度,RT-PCR及Western blot检测T24细胞内PLCε的表达情况。结果酶切及测序鉴定证实pAdTrack-U6-PLCε及重组pAdEasy-U6-PLCε质粒构建正确,并成功转染至HEK-293细胞,重组腺病毒Ad-U6-PLCε滴度达1.5×1012,经重组腺病毒感染的T24细胞内PLCεmRNA及蛋白表达均明显降低(P<0.05)。结论已成功构建针对PLCε基因的shRNA重组腺病毒载体Ad-U6-PLCε,为应用基因沉默技术研究PLCε对膀胱癌发生发展的作用奠定了基础。
Objective To construct a recombinant adenovirus vector expressing green fluorescent protein (GFP) tag PLCε gene Ad-U6-PLCε, which laid the foundation for the gene therapy of bladder cancer by using gene silencing technology. Methods The expression vector pGenesil-PLCε and the negative control plasmid pGenesil-NP expression promoter U6 and shRNA were cloned into the shuttle plasmid pAdTrack with enhanced green fluorescent protein (EGFP) reporter gene. After identification by restriction enzyme and sequencing, The recombinant shuttle plasmid was linearized by PmeI and transformed into competent AdEasier. Recombinant pAdEasy-U6-PLCε plasmid was linearized with PacI and transfected into HEK-293 cells. The recombinant adenovirus Ad-U6-PLCε was packaged and amplified. The titer was determined by RT-PCR and Western blot. PLCε expression. Results Restriction endonuclease digestion and sequencing confirmed that pAdTrack-U6-PLCε and recombinant pAdEasy-U6-PLCε plasmid were correctly constructed and successfully transfected into HEK-293 cells. The recombinant adenovirus Ad-U6-PLCε titer was 1.5 × 1012, The PLCεmRNA and protein expression in adenovirus infected T24 cells were significantly decreased (P <0.05). Conclusion Ad-U6-PLCε, a recombinant adenovirus vector targeting PLCε gene, has been successfully constructed and laid the foundation for the study of PLCε on the occurrence and development of bladder cancer using gene silencing technology.