过氧化物酶增殖子活化受体-γsiRNA腺病毒载体的构建

Construction of pAdeno-siPPARγ

目的:构建针对过氧化物酶体增殖子活化受体-γ(PPARγ)基因的siRNA腺病毒载体。方法:依据siR-NA设计原则,在PPARγmRNA(AF013266)序列中设计2个特异性靶序列(36-54、73-91),体外合成对应发卡样DNAs,退火后先将其克隆入pSIREN-Shuttle载体,再亚克隆入pAdeno腺病毒载体,对插入序列进行DNA序列分析。结果:发卡样siRNA单链DNA寡核苷酸退火后,电泳可见明亮靶条带。连接退火寡核苷酸和pSIREN-Shuttle载体得到阳性克隆pSIREN-Shuttle-siPPARγ-36、pSIREN-Shuttle-siPPARγ-73。同源重组后得到亚克隆pAdeno-siPPARγ-36、pAdeno-siPPARγ-73,测序证实插入序列与设计序列完全一致。结论:成功构建2个靶向PPARγ基因的siRNA载体pAdeno-siPPARγ-36和pAdeno-siPPARγ-73。

Aim： To construct the siRNA adenovirus vector targeting peroxisome proliferator-activated receptor-γ （PPARγ）. Methods：Two specificity target sequences（ 36-54.73-91 from PPARγ mRNA sequencer AF013266 were designed according to the principle of design on siRNA, then the corresponding hairpin-shaped DNA fragment was synthesized in vitro. The product was cloned into pSIREN-Shuttle vector after annealing,then subeloned into siRNA adenovirus vector pAdeno. The insertion DNA sequences were analyzed. Results ： The hairpin-shaped siRNA single strand DNA oligonucleotide showed an obvious electrophoresis strip after annealing. The positive clones pSIREN-Shuttle-siPPARγ-36 and pSIREN-Shuttle-siPPARγ-73 were obtained by connecting the annealed oligonucleotide with pSIREN-Shuttle vector. Positive subclone pAdeno-siPPARγ-36 and pAdeno-siPPARγ-73 were obtained by homologous recombination. The inserted sequences were confirmed by sequencing. Conclusion： Two siRNA adenovirus vector targeting PPARγ,pAdeno-siPPARγ-36 and pAdeno-siPPARγ-73, are successfully constructed.