目的：构建DEK基因的RNA干扰（ RNAi）慢病毒表达载体，并对其生物学功能进行初步检测。方法采用RNAi技术，根据DEK基因的序列，确定其有效靶序列，合成DEK基因的Oligo DNA，退火形成双链DNA，将其克隆到经BamHⅠ与EcoRⅠ酶切后的小干扰RNA（ siRNA）表达载体PSIH-H1上，产生PSIH-H1-DEK慢病毒载体，筛选阳性克隆并测序鉴定。与3个包装质粒共转染293T细胞，包装成慢病毒后感染乳腺癌细胞ZR75-1，经嘌呤霉素（ puromycin，puro）筛选2周后，收集部分细胞利用实时聚合酶链反应（ real time-PCR，RT-PCR）和Western印迹分别检验DEK在信使RNA （ mRNA ）和蛋白水平的敲低效果，并通过细胞生长实验检测DEK对人乳腺癌细胞系ZR75-1细胞生长的影响。结果 PCR和DNA测序结果证实，DEK siRNA慢病毒表达载体PSIH-H1-DEK构建成功。 RT-PCR和Western印迹结果显示，构建的DEK siRNA可有效抑制DEK基因的表达，并由此建立了敲低DEK的稳定克隆。生长曲线实验表明， DEK siRNA可抑制人乳腺癌细胞系ZR75-1细胞的生长。结论成功构建了DEK基因的RNAi慢病毒表达载体，感染人乳腺癌细胞系ZR75-1细胞后，有效沉默了ZR75-1细胞中的DEK基因的表达，为进一步研究DEK基因在乳腺癌中的作用奠定基础。%Objective To construct the lentiviral vector of RNA interference(RNAi) for DEK,and to detect its effect on breast cancer cell growth.Methods The DEK siRNA was designed and constructed based on DEK sequence using a lentiviral vector.The lentivirul vector containing DEK siRNA was named PSIH-H1-DEK as confirmed by PCR and sequenceing.PSIH-H1-DEK was then packaged with accessory plasmids into lentivirus in 293T cells and selected for 2 weeks with puromycin ( puro ) before the mixed colonies stably expressing DEK siRNA were obtained and the DEK expression was detected by real time PCR( RT-PCR) and Western blotting.The effect of DEK siRNA on ZR75-1 cell growth was determined by cell counting kit.Results Western blot and RT-PCR showed that PSIH-H1-DEK siRNA could suppress DEK gene expression.Suppression of DEK could markedly inhibit the growth of ZR75-1 cells.Conclusion The lentivirus-mediated DEK siRNA is obtained,which will facilitate further research on DEK function in breast cancer development.
展开▼
