逆转录病毒载体介导的RNAi抑制乳腺癌细胞BSP基因表达

目的构建抑制人日5P基因表达的RNA干扰逆转录病毒载体，筛选建立稳定抑制BSP表达的MDA-MB-231BO细胞系。方法构建靶向人BSP基因的逆转录病毒重组质粒pSilencer—BSP27、pSilencer—BSP81和pSilencer—BSP100。将其包装成病毒后感染MDA—MB-23180细胞。用RT—PCR和Western印迹检测成功感染细胞（231BO．BSP27、231BO．BSP81、231BO—BSP100细胞）BSP基因的抑制效果。结果双酶切和测序结果显示重组核酸片段序列与设计的序列完全相同。RT—PCR和Western印迹结果显示231BO—BSP27、BO—BSP81和231BO—BSP100细胞BSP基因在mRNA水平和蛋白水平抑制效率分别为：70．8％、79．4％和30．1％：69．3％，75．2％和27．8％。结论成功构建抑制人BSP基因表达的RNAi逆转录病毒载体pSilencer．BSP27、pSilencer．BSP81和pSilencer—BSP100。获得高效稳定抑制BSP表达的231BO—BSP27、231BO．BSP81细胞系。

Inhibition of BSP Gene Expression in Breast Cancer Cells by Retrovirally Mediated RNAi

Objective To construct RNA interfering retroviral vectors effectively suppressing BSP gene expression and establish MDA-MB-231BO cell line stably displaying inhibition of BSP gene expression. Methods The designed fragments targeting human BSP gene were ligated into pSilencer^TM5, 1-U6 Retro-Vector. Three resultant retroviral recombinant plasmids were obtained, including pSilencer-BSP27, pSilencer-BSP81 and pSilencer-BSP100. Double digestion and DNA sequencing were performed to identify each recombinant plasmid. Each recombinant plasmid was then applied to transfect virus packaging 293 cells to produce virus suspensions. Following infection of MDAMB-231BO cells with virus suspensions, 231BO-BSP27, 231BO-BSP81 and 231BO-BSP100 cell lines were selected and established. The inhibitive effect of BSP gene expression in each cell line was determined by RT-PCR and Western Blot. Results Double restriction enzyme digestion and DNA sequencing results proved that the three recombinant plasmids were correctly constructed. RT-PCR and Western Blot results confirmed that BSP mRNA level and protein expression were reduced to 70. 8% , 79.4% and 30. 1% , and 69.3% , 75.2% and 27. 8% in 231BO-BSP27, 231BO- BSP81 and 231BO-BSP100 cell lines, respectively. Conclusion RNA interfering retroviral vectors pSilencer-BSP27, pSilencer-BSP81 and pSileneer-BSP100 were successfully constructed with inhibitive effect on human BSP gene expression. The obtained 231 BO-BSP27 and 231 BO-BSP81 cell lines showed stably and remarkably reduced BSP expression at both mRNA and protein level.