p300基因小干扰RNA慢病毒载体的构建及其对乳腺癌ZR75-1细胞的影响  

Lentiviral Vector for p300 Small-Interfering RNA and its Effect on Breast Cancer ZR75-1 Cell Growth

作　　者：陈志达[1] 丁丽华[2] 张亚楠[2] 刘婕[2] 朱杰[2] 叶棋浓[2] 卫勃[1] 

机构地区：[1]解放军总医院普通外科,北京100853 [2]军事医学科学院生物工程研究所,北京100850

出　　处：《生物技术通讯》2014年第5期621-624,共4页Letters in Biotechnology

基　　金：国家自然科学基金(81101883;81272231);北京市科技新星计划(2009A38;2011112)

摘　　要：目的:构建p300基因的小干扰RNA(siRNA)慢病毒表达载体,检测其对p300蛋白表达的干扰,以及对乳腺癌细胞ZR75-1生长的影响。方法:利用RNA干扰(RNAi)技术设计并合成针对p300基因的siRNA,并将其克隆到siRNA表达载体pSIH-H1上,经酶切和测序验证,用293T人胚肾细胞包装p300 siRNA慢病毒,感染乳腺癌细胞ZR75-1,建立敲低p300表达的稳定细胞株,通过实时定量PCR和Western印迹检验RNAi的干扰效果,利用细胞生长实验检测p300 siRNA对ZR75-1细胞生长的影响。结果:酶切和测序证明构建了p300 siRNA真核表达载体;实时定量PCR和Western印迹证明构建的siRNA能有效抑制p300基因的表达,并建立了敲低p300表达的稳定细胞株;细胞生长实验证实p300 siRNA可有效促进ZR75-1细胞的生长。结论:构建了p300基因的siRNA真核表达载体,转染细胞后能有效抑制p300基因的表达,且能促进ZR75-1细胞的生长,表明构建的p300 siRNA具有功能。Objective： To construct the lentiviral vector for p300 small-interfering RNA（siRNA）, and to detect its effect on breast cancer ZR75-1 cell growth. Methods： The p300 siRNA was designed and constructed based on human p300 cDNA sequence using a lentiviral vector. After enzyme digestion and sequencing, pSIH-HI-p300 siRNA was then packaged with accessary plasmids into lentivirus in 293T cells. After infected with lentivirus and selected by puromycin, mixed ZR75-1 colonies stably expressing p300 siRNA were obtained and the fusion p300 expression was detected by real-time quantification PCR（qRT-PCR） and Western blot. Finally, the effect of p300 siRNA on ZR75-1 growth was determined by cell counting kit. Results： Western blot and qRT-PCR showed that pSIH-HI-p300 siRNA could suppress the p300 gene expression. Suppression of p300 could markedly promote the growth of ZR75-1 cells. Conclusion： The lentivirus-mediated p300 siRNA was obtained, which lays the foundation for further research on p300 signaling pathway.

关 键 词：p300基因 小干扰RNA 慢病毒载体 

分 类 号：Q78[生物学—分子生物学]