慢病毒GV115-caspase-3 siRNA重组表达系统的构建及鉴定

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。

Construction and Detection of GV115-caspase-3 siRNA.

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-caspase-3 siRNA was constructed by gene synthesis and subclone technique. The GV115-caspase-3 siRNA was detected by PCR and DNA sequencing. After the lentivirus had been packaged, the 293T cells were transfected by GV115-caspase-3 siRNA. The translation of caspase-3 gene transfected by GV115-caspase3 siRNA was detected using 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.