靶向过氧化物酶体增殖子活化受体-γ基因siRNA腺病毒载体的构建与鉴定  

Construction and identification of siRNA adenovirus vector to target PPARγ gene

作　　者：王义生[1] 王少华[1] 李月白[2] 赵国强[3] 张弛[1] 李振伟[1] 殷力[1] 刘宏建[1] 

机构地区：[1]郑州大学第一附属医院骨科河南省高等临床医学重点学科开放实验室,450052 [2]郑州大学基础医学院生物化学与分子生物学教研室 [3]郑州大学基础医学院微生物免疫教研室

出　　处：《中华实验外科杂志》2008年第5期656-657,共2页Chinese Journal of Experimental Surgery

基　　金：国家自然科学基金资助项目（30672128）

摘　　要：目的构建、鉴定靶向过氧化物酶体增殖子活化受体-γ（PPAR-γ）基因的siRNA腺病毒载体。方法遵循siRNA片段的设计原则，依据GenBank中PPAR吖基因（AF013266）的序列，设计3个特异性靶序列（36～54位、73～91位和404～422位）；体外合成对应发卡样DNA片段，经退火后，将其克隆入pSIREN．Shuttle载体，通过PCR和I-CeuI和PI-SceI酶切鉴定后，再亚克隆入pAdeno-X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；PCR扩增和酶切鉴定得到阳性克隆pSIREN-Shuttle—siPPAR-γ1、pSIREN-Shuttle—siPPARγ-2和pSIREN-Shuttle—siPPARγ-3。亚克隆重组腺病毒载体pAdeno—X．siPPAR^t-1、pAdeno—X-siPPARγ-2和pAdenc-X—siPPARγ-3，经测序鉴定证实插入DNA序列与设计完全一致。结论成功构建3个靶向PPARγ基因的siRNA重组腺病毒载体，为进一步的基因治疗研究奠定了实验基础。Objective To Construct and identify the target siRNA adenovirus vector to peroxisome pmliferator-activated receptor-γ （PPARγ）. Methods Three specificity target sequences （36-54nt, 73-91nt and 404--422nt） were designed from PPARγ mRNA sequence （AF013266） according to the principle of design on siRNA,the corresponding hairpin-shaped DNA fragments synthesized in vitro. After annealing, the shRNA DNAs （product） were cloned into pSIREN-Shuttle vector, which identified by PCR and digestion with I-Ceu I and PI-Sce I, then subcloned into adenovirus vector pAdeno-X for expressing shRNA. The insertion DNA sequences were analyzed. Results Hairpin-shaped siRNA single strand oligonucleotide showed the obvious electrophoresis strip after annealing. The positive clone pSIREN-ShuttlesiPPARγ was obtained, which was confirmed correct through PCR amplification identification and sequencing. Recombinant adenovirus vectors pAdeno-X-siPPARγ-l, pAdeno-X-siPPARγ-2 and pAdeno-X- siPPARγ-3 were obtained and identified by sequencing. The inserted sequences were confirmed to be iden- tical with the designed sequences. Conclusion Recombinant adenovirus vector pAdeno-X-siPPARγ had been constructed successfully,which can be used for further investigation of gene therapy.

关 键 词：RNA干扰 过氧化物酶体增殖子活化受体-γ 腺病毒载体 

分 类 号：R346[医药卫生—基础医学]