EZH2基因RNAi慢病毒载体的包装及鉴定

目的 外周T细胞淋巴瘤（PTCL）源于成熟的胸腺后淋巴细胞,是一类少见的生物学行为及临床表现均高度异质性的疾病,且预后差.包装zeste基因增强子同源物2（EZH2）RNA干扰（RNAi）慢病毒载体,可为今后相关实验研究奠定基础.方法 靶向EZH2基因的短发夹RNA（EZH2-shRNA）质粒转化感受态细菌,筛选阳性克隆,经测序鉴定正确后通过脂质体将慢病毒三质粒系统共转染293T细胞,进行慢病毒包装.病毒载体感染Hut78细胞后,观察感染效率,并用反转录定量PCR（RT-qPCR）和Western Blot检测EZH2mRNA和蛋白表达水平.结果 成功包装了慢病毒表达载体,对Hut78细胞的感染效率在95％以上.RT-qPCR和Western Blot检测结果显示,EZH2基因在靶细胞中的表达水平降低.结论 成功包装并鉴定了EZH2基因RNAi慢病毒表达载体.

Package and identification of a lentiviral vector of RNA interference containing EZH2 gene

Objective Peripheral T-cell lymphomas （PTCLs） originated from mature,post thymic T cells are a rare heterogeneous group of clinically aggressive non-Hodgkin lymphoma （NHL） with a dismal prognosis.This study was aimed to package and identify a lentiviral vector of RNA interference （RNAi） targeting enhancer of zeste homolog 2 （EZH2）,would lay the foundation for the future study.Methods After transformation into competent E.coli bacteria,the candidate clones were identified by DNA sequencing.The EZH2-short hairpin RNA （shRNA） plasmid and the two packaging plasmids were co-transfected into the human embryonic kidney 293T cells by lipofectamine 2000 to produce the lentiviral vector.The Hut78 cells were infected with the lentiviral vector obtained and the transfection efficiency was assessed under the fluorescent microscope.Then the expression of EZH2 in the transfected cells was determined by RT-qPCR and Western Blot.Results The lentiviral RNAi vector for the EZH2 gene was packaged successfully.Strong red fluorescence was observed in the Hut78 cells under the fluorescent microscope after co-transfection of the cells with the 3 plasmids of the lentiviral vector.The transfection efficiency of the collected virus exceeded 95％ in the Hut78 cells.The lentiviral vector significantly inhibited the expression of EZH2 at both the mRNA and protein levels.Conclusions The lentiviral RNAi vector of EZH2 has been successfully packaged and identified.