摘要
目的:构建Rabex-5 RNAi慢病毒表达载体,并转染人乳腺癌细胞株MCF-7,观察其对Rabex-5基因的沉默效应。方法:将设计合成的单链引物退火成双链oligo序列,连接入经AgeⅠ和EcoRⅠ双酶切线性化的pMAGic慢病毒质粒载体中,经转化DH5α感受态细胞并筛选出阳性克隆,测序鉴定。用293FT细胞包装产生慢病毒,感染MCF-7细胞,利用Real-time PCR和Western blot鉴定抑制Rabex-5表达的效果。结果:PCR与测序鉴定证实成功构建了Rabex-5 RNAi的慢病毒载体,转染MCF-7细胞后,与未干扰组和阴性对照组相比,Rabex-5的mRNA和蛋白表达下调,以RNAi3的沉默效应最佳。结论:成功构建了Rabex-5 RNAi慢病毒表达载体,其可使MCF-7细胞株Rabex-5表达下调,为进一步研究靶向Rabex-5 RNAi对乳腺癌细胞恶性生物学行为变化及基因治疗研究奠定了实验基础。
Objective：To construct a lentiviral vector for RNA interference（RNAi） of Rabex-5 gene and to detect its silence effect on Rabex-5 in MCF-7 cells.Methods：The synthesised single-stranded primer was annealed to double-stranded oligo sequences and sub-cloned into linear pMAGic lentiviral plasmid vector digested by enzyme AgeⅠ and EcoRⅠ.DH5α competent cells were transformed and positive clone were screened,identified and sequenced.The recombinant lentivirus was packaged into mature lentivirus by 293FT cells and used to infect MCF-7 cells.The expression of Rabex-5 in the cells was detected by real-time PCR and Western blot.Results：PCR and sequencing verified that the recombinant lentivirus plasmid Rabex5-shRNA was successfully constructed.Compared with the negative control and the blank control cells,Rabex-5 mRNA and protein level was reduced in MCF-7 cells infected by lentivirus.RNAi3 had the best effect of gene silence.Conclusion：The lentiviral RNAi expression vector targeting Rabex-5 gene is successfully constructed and it can downregulate the expression of Rabex-5 in MCF-7 cells,which lays the experimental foundation for the research on the changes of malignant biological activity of tumor cell lines and gene therapy.
出处
《重庆医科大学学报》
CAS
CSCD
北大核心
2012年第1期34-38,共5页
Journal of Chongqing Medical University
基金
重庆市教委基金资助项目(编号:KJ100317)
