p100蛋白慢病毒干扰载体及p100沉默的HepG2稳定细胞系的构建.pdfVIP

—墨连堕垫垫!!!!旦篁塑堂笙!塑 529 i“n．0153—9896．2012．06．001 fi：10．3％9，j 论著 稳定细胞系的构建+ 黄丽尹洁丁建民宋娟高星杰经翔杨洁6 摘要目的：用表达shRNA的干扰慢病毒载体系统构建p100基因稳定沉默的HepG2人肝癌细胞系。方法：通过 DNA shRNA表达质粒与慢病毒包装质粒 并构建pLKO．3G-p1001慢病毒载体，用双酶切及测序进行鉴定。将构建好的p100 共转染至包装细胞293T，包装产生病毒颗粒。运用病毒颗村感染HepG2细胞，获得p100稳定沉默的细胞系。荧光显 果的慢病毒载体，并获得了稳定沉默p100及对照的HepG2细胞株。慢病毒感染HepG2细胞后．荧光显微镜卜观察列 90％以上细胞发出绿色荧光，Western 功构建了具有p100干扰效果的慢病毒干扰载体及p1【)o稳定沉默的HepG2细胞系。 关键词RNA干扰细胞系，肿瘤质粒慢病毒载体p100蛋白HepG2细胞系 Constructionof 00KnockDownStable CelILinewitha 00 P1 HepG2 pl shRNA LentiviraI ExpressionSystem HUANGLi，YINJie，DINGlianmin，SONGJuan，GAO Jie Xingjie，JINGXiang，YANG Medicsf 300070．China DepartmentofImmunology,rianjinUniversity,rianjin Abstract constructalentiviralvector ashorl thefunc· Objective：To expressinghairpinRNA(shRNA)．whichtargets tional andtoconstructa stableknock-downedcelllinewiththis shRNA lentiviral p100gene p100 HepG2 p100 expressing double—strandedshRNA the W5 andclonedintothe system．Methods：A targetingp100gene designed，synthesized vector．Positivecloneswereverifiedwithdouble and thevalidconstructions pLKO．3G enzymedigestionsequencing．Then werecotransfectedwiththeother2packaginginto2931cellsto lentiviral shRNA plasmids packa