生长抑素基因靶向siRNA表达载体的构建及对生长抑素基因表达的抑制作用  

Construction siRNA expression vector targeting somatostatin and its inhibition on somatostatin expression in vitro

作　　者：吴琼[1] 张永亮[1] 赵志辉[1] 刘宇鹏[1] 周立光[1] 任晓慧[1] 侯锋[1] 章倩倩[1] 吕铁钢[1] 郝林琳[1] 刘松财[1] 

机构地区：[1]吉林大学畜牧兽医学院,吉林长春130062

出　　处：《中国兽医学报》2008年第3期276-280,共5页Chinese Journal of Veterinary Science

基　　金：国家自然科学基金资助项目(30570973);吉林大学农学部青年科研基金资助项目

摘　　要：合成含靶向生长抑素(Somatostatin,SS)前体基因的siRNA转录模板的发夹结构,插入pSilencerTM2.1-U6载体构建重组质粒2.1-S。提取小鼠下丘脑组织总RNA,PCR扩增获得SS前体基因cDNA,构建真核表达载体pcDNA3.1-SS、pEGFP-SS。将2.1-S分别与pcDNA3.1-SS、pEGFP-SS共转染入细胞中瞬时表达,检测SS前体基因表达变化。结果显示,重组质粒在E.coli(DH5α)内扩增,酶切及测序鉴定证明siRNA转录模板完整,正确地插入到pSilencerTM2.1-U6质粒中,重组质粒pcDNA3.1-SS与pEGFP-SS经测序序列完全正确。转染细胞在mRNA水平及蛋白水平均检测到2.1-S对SS前体基因表达的抑制。这表明,SS基因靶向siRNA稳定表达载体能在哺乳动物细胞中表达,并对靶基因呈抑制作用。To construct a stable expression vector of siRNA targeting mouse somatostatin（SS） and test its inhibition on transfected SS expression in cells,a hairpin structure of siRNA transcript template targeting SS gene was synthesized and cloned to pSilencerTM 2.1-U6.The constructed siRNA vector was cotransfected into cells with SS expression vector（pcDNA3.1-SS or pEGFP-SS）.The SS expression alteration was detected via RT-PCR or EFGP fluorescence observation.The results showed a siRNA expression vector targeting SS,pSilencerTM 2.1-U6-SS,was constructed successfully.shRNA expressed by pSilencerTM 2.1-U6-SS revealed strong inhibition on SS expression in vitro,proved by great decrease in mRNA level and EGFP flourescence.It is concluded that the siRNA expression vector has been constructed successfully and enables to exert suppression on SS gene expression in vitro.

关 键 词：生长抑素 SIRNA 稳定表达载体 

分 类 号：S852.2[农业科学—基础兽医学] Q78[农业科学—兽医学]