EZH2基因3'非翻译区慢病毒载体构建及重组体筛选

摘要

Objective To construct lentiviral vector carrying human EZH2 3' untranslated region (UTR) and maintain its stable integration of recombinant in MCF-7 cells of breast cancer,and to lay the foundation for the further screening of MicroRNAs (miRNAs).Methods The EZH2 3'UTR was extracted and amplified by real-time fluorescence quantitation polymerase chain reaction (RT-PCR),and was constructed into recombinant lentiviral vector,CTX-lentivirus-EZH2 3'UTR vector.The 293T cells were contransfected with the recombinant lentiviral vector together with lentivirus package plasmid to produce lentiviral particles by liposome method.Cracking liquid cracked virus particles,and virus titer was measured according to the expression level of CMV promoter.MCF-7 ceils were infected with recombinant lentiviral vector.Puromycin was used to screen the stable MCF-7 cells.The RT-PCR and Western blotting were determined the integration of EZH2 3'UTR into MCF-7 cells.Results EZH2 3'UTR was cloned in lentiviral vector.By RT-PCR,the virus titer reached to 4.3 × 109 copyies/mL.The optimal concentration for screening stable integration of MCF-7 cells was 0.2 μg/mL.In infected MCF-7 cells,by RT-PCR and Western blotting,the EZH2 3' UTR mRNA was integrated into MCF-7 genome.The difference between the experimental group and the control group was significant(P ＜ 0.01).Conclusions EZH2 3'UTR lentiviral vector has successfully constructed and maintained stable integration in MCF-7 cells of breast cancer.%目的 构建携带人EZH2基因3’非翻译区(3'UTR)的慢病毒筛选载体并实现该重组体在人乳腺癌MCF-7中稳定整合,为下一步MicroRNAs(miRNAs)筛选奠定基础.方法 从组织中提取并用实时荧光定量聚合酶链反应(RT-PCR)扩增出EZH2基因3'UTR,构建携带EZH2基因3'UTR的重组慢病毒载体CTX-Lentivirus-EZH2 3'UTR,脂质体法将重组的慢病毒载体和包装载体混合物共转染工具细胞293T细胞,包装产生慢病毒颗粒.用裂解液裂解病毒颗粒,根据慢病毒载体上的CMV启动子表达量检测病毒滴度.用慢病毒颗粒感染乳腺癌MCF-7细胞后,通过嘌呤霉素筛选稳定整合的乳腺癌细胞MCF-7,用Real-Time PCR和Western blotting方法检测EZH2基因3'UTR是否整合入细胞MCF-7中.结果 PCR扩增检测阳性菌落和测序结果证实,EZH2基因3'UTR成功定向克隆到慢病毒载体上.Real-Time PCR检测病毒滴度为4.3 × 109拷贝/mL.筛选稳定整合MCF-7细胞的最佳浓度为0.2μg/mL.重组慢病毒转导MCF-7后,Real-Time PCR和Western blotting方法分别检测EZH2基因3'UTR整合到乳腺癌细胞MCF-7基因组中.慢病毒感染实验组和对照组比较,差异有统计学意义(P＜0.01).结论 成功构建携带EZH2基因3'UTR慢病毒筛选载体,实现重组体在乳腺癌MCF-7细胞中稳定整合.