摘要
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 the recombinant adenovirus vector for expression of shRNA targeting phospholipase C epsilon（PLCε） gene,with a green fluorescent protein（GFP） tag,and lay a foundation of gene therapy of bladder cancer by gene silencing technique.Methods The U6 expression promoter and shRNA sequences of interfering plasmid pGenesil-PLCε and negative control plasmid pGenesil-NP were cloned to shuttle plasmid pAdTrack with enhanced GFP（EGFP） report gene.The constructed recombinant shuttle plasmid pAdTrack-U6-PLCε was identified by restriction analysis and sequencing,then linearized with PmeⅠand transformed to competent AdEasier.The constructed recombinant plasmid pAdEasy-U6-PLCε was linearized with PacⅠ and transfected to HEK-293 cells for packaging.The obtained recombinant adenovirus was propagated in a large quantity and determined for titer.The expressions of PLCε in T24 cells were determined by RT-PCR and Western blot.Results Restriction analysis and sequencing proved that recombinant plasmids pAdTrack-U6-PLCε and pAdEasy-U6-PLCε were constructed correctly and transfected to HEK-293 cells successfully.The titer of recombinant adenovirus was 1.5×1012.Both the expression levels of PLCε mRNA and protein in T24 cells infected with the recombinant adenovirus decreased significantly（P 0.05）.Conclusion The recombin antadenovirus vector for expression of shRNA targeting PLCε gene was successfully constructed,which laid a foundation of study on role of PLCε in ongoing of bladder cancer by gene silencing technique.
出处
《中国生物制品学杂志》
CAS
CSCD
2013年第1期51-55,共5页
Chinese Journal of Biologicals
基金
国家自然科学基金(81072086)
