应用珠蛋白启动子驱动荧光蛋白在K562细胞中表达 作者:谢书阳王萍玉姚宝洪焦飞王桂兰杨成 【关键词】红细胞系特异性启动子; 荧光蛋白; 基因表达 【Abstract】 Objective Studing the effect of erythroid-specific promoter can effectively drive expression of green/red fluorescence protein(GFP/RFP) in K562 cells. Methods Erythroid-specific promoter(β globin promoter) was amplified by PCR from human genome DNA, which was cloned into pcDNA vector to drive GFP/RFP gene expression. Then the vectors were transfected into K562 cells to prove the effect of the promoter by using fluorescence microscopy and RT- Fluorescence microscopy showed that GFP/RFP can strongly express in K562 cells. And the mRNA was also shown to express effectively by RT-PCR The erythroid-specific promoter can obviously drive gene expression in K562 cells, which is valuable for gene therapy to leukemia as well as other hematology diseases. 【Key words】 erythroid-specific promoter,fluorescence protein,gene expression 人类珠蛋白基因是一种研究人类基因组结构与功能相关性的理想材料。珠蛋白基因的表达,具有发育阶段特异性和珠蛋白表达的组织特异性[1]。而启动子在保证珠蛋白各功能基因组织及发育阶段特异性表达方面起重要作用[2]。因此克隆 β-珠蛋白启动子对血液病的治疗和基因表达调控研究具有重要价值。本实验旨在克隆β-珠蛋白启动子,用其驱动目的基因在造血细胞系中表达,验证启动子的有效性,为血液病基因治疗作基础准备。 1 材料与方法 材料脂质体2000购自Invitrogen 公司,RNA 提取试剂盒购自U-GENE公司,MluI、NheI、SpeI等酶、逆转录酶购自TaKaRa公司,K562细胞株购自中科院上海细胞生物研究所,DMEM培养基、胎牛血清购自GIBCO公司,GFP和RFP载体由山东大学崔行教授惠赠。 启动子的扩增从人的基因组DNA中扩增β珠蛋白启动子,引物序列正向:5′-GCAGAACACT-3′,反向:5′-TCA-3′;PCR反应条件为:94℃ 3
