摘要
目的构建高效的慢病毒GV115-caspase-3 siRNA重组表达系统。方法根据RNA干扰序列设计原则,设计4个可能的caspase-3 siRNA序列。应用全基因合成技术和亚克隆技术构建GV115-caspase-3 siRNA并采用PCR和测序对其鉴定。病毒包装后转染人胚肾293T细胞,通过应用RT-PCR技术检测转染后caspase-3基因敲减效率,筛选高效的GV115-caspase-3 siRNA。结果 GV115-caspase-3 siRNA质粒PCR鉴定显示位于341bp附近的条带,测序结果与设计的基因序列完全吻合,4个可能的caspase-3 siRNA序列中基因敲减效率最高的可达到84.2%。结论成功构建高效的慢病毒GV115-caspase-3 siRNA重组表达系统。
Objective To construct and detect the GV115 - caspase - 3 siRNA. Methods On the basis of RNAi design principle, four caspase -3 siRNA sequence were designed. GV115 -easpase- 3 siRNA was constructed by gene synthesis and subelone technique. The GV115 - easpase -3 siRNA was detected by PCR and DNA sequencing. After the lentivirus had been packaged, the 293T cells were transfected by GVll5 -caspase- 3 siRNA. The translation of caspase -3 gene transfected by GVll5 -caspase3 siRNA was detected u- sing RT- PCR and the most effective GV115 -caspase- 3 siRNA was screened. Results The GV115 -caspase -3 siRNA was proved to be right using PCR and DNA sequencing. The gene knocking rate of the most efficient GV115 - caspase - 3 siRNA was 84.2%. Conclusion The GV115 -caspase- 3 siRNA was constructed successfully.
出处
《医学研究杂志》
2015年第11期40-43,共4页
Journal of Medical Research
基金
国家自然科学基金资助项目(81171758)
