人EZH2基因编码区及3'非翻译区融合蛋白重组慢病毒表达载体构建及其在293T细胞中的表达  

Construction of a recombination lentiviral vector carrying EZH2 gene coding region and 3'untranslated region fusion protein and its expression in 293T cells

作　　者：彭攸[1] 赵卫卫[1] 李晓娇[1] 王玉平[1] 卢晓佳[1] 邬美玉[2] 冯景[1] 

机构地区：[1]南方医科大学附属奉贤医院检验科,上海201400 [2]南方医科大学附属奉贤医院妇产科,上海201400

出　　处：《中华临床医师杂志（电子版）》2012年第24期8151-8155,共5页Chinese Journal of Clinicians(Electronic Edition)

基　　金：国家自然科学基金青年科学基金项目(81202104);上海市自然科学基金面上项目(12ZR1426300)

摘　　要：目的构建携带人EZH2基因编码区(coding region,CDS)及3'非翻译区(3'untranslated region,3'UTR)融合红色荧光蛋白(red fluorescent protein,RFP)重组慢病毒表达载体,使mCherry-EZH2融合蛋白在293T细胞中稳定表达。方法运用In-Fusion定向克隆技术构建人EZH2基因CDS区及3'UTR融合蛋白重组慢病毒表达载体,测序无误后抽提质粒,转染293T细胞,包装病毒,测定病毒滴度。Western blot检测mCherry-EZH2的表达。结果测序证实重组慢病毒表达载体基因序列完全正确;病毒颗粒包装成功,滴度为1.59×109IU/ml;Western blot检测293T细胞中可以表达mCherry-EZH2融合蛋白。结论成功构建人EZH2基因CDS区及3'UTR融合蛋白重组慢病毒表达载体及病毒,其mCherry-EZH2可以在293T细胞中稳定表达。为进一步分析可能调控EZH2基因CDS区及3'UTR表达的miRNA奠定前期实验基础。Objective To construct a recombination lentiviral vector carrying human EZH2 gene coding region and 3’UTR fusion protein and its expression in 293T cells.Methods The lentiviral vector was made by the In-Fusion technology.The lentiviral expression vector was obtained after screening followed by sequencing.The lentiviral vector was used to transfect 293T cells and package virus,and the virus titers were determined.Western blotting was used to detect the expression of mCherry-EZH2 in 293T cells.Results The lentiviral vector was successfully constructed,which was confirmed by the assessment of sequencing.After the vector transfected into 293T cells,the lentiviral particles were successfully packaged.Viral titer was 1.59×109 IU/ml.Western blotting was confirmed that the mCherry-EZH2 expressed in 293T cells.Conclusions The lentiviral expression vector was successfully constructed,which expressed the mCherry-EZH2 in 293T cells stablely.

关 键 词：3’非翻译区 EZH2基因 红色荧光蛋白 慢病毒载体 

分 类 号：R730.2[医药卫生—肿瘤]