慢病毒介导的小鼠生长抑素shRNA序列的设计和筛选  

Design and screening of lentiviral-mediated shRNA sequences targeting mouse somatostatin

作　　者：郝林琳[1] 于浩[1] 李莉[1] 王博[1] 王佳[1] 刘松财[1] 张永亮[1,2] 

机构地区：[1]吉林大学畜牧兽医学院,吉林长春130062 [2]华南农业大学动物科学学院,广东广州510643

出　　处：《中国兽医学报》2011年第1期65-69,共5页Chinese Journal of Veterinary Science

基　　金：国家自然科学基金资助项目(30771572);国家自然科学基金联合基金资助项目(u0731004)

摘　　要：生长抑素(SS)是一种多功能的脑肠肽,在动物生长轴中主要作为生长激素(GH)的负性调控因子。本研究通过慢病毒表达质粒介导shRNA,瞬时转染自身表达SS的BHK-21细胞,筛选出了靶向小鼠SS的最有效的siRNA序列。首先,分别在小鼠SS基因mRNA的246、433和539bp处找到潜在靶位点,合成3条siRNA转录模板的发夹结构以及1条阴性对照,体外退火后插入pshRNA-copGFP lentivector构建重组质粒。然后,通过对转染BHK-21细胞的荧光观察、Real-time PCR、免疫组化以及RIA等检测,转染细胞SS在mRNA水平及蛋白水平均检测到不同程度的抑制(P<0.05)。其中,psh2(SS 433～451)表现出了最高的沉默效率(转染效率68.9%时,mRNA水平的沉默效率达59.3%,P<0.05;蛋白质水平的沉默效率达55.6%,P<0.05)。本研究为降低SS在动物体内的分泌,相应提高GH的浓度,进而为促进动物生长的研究奠定了基础。As a multifunctional braingut petide,Somatostatin（SS） is an important negative regulator of Growth hormone（GH） in the GH-IGF-1 growth axis.In this study,we screened the best siRNA sequence targeting mouse SS by the transient-transfection of the lentiviral mediated shRNA into BHK-21 cells which secrete SS themselves.First,three potential sites in mouse SS mRNA sequence（start from 246,433,539 bp,seperately） were selected and synthesized,as well as one negative shRNA.After annealing in vitro,four of pshRNA-copGFP lentivectors were reconstructed.Then,the lentiviral-shRNAs were transient-transfected into BHK-21 cells,both SS mRNA and protein levels were down-regulated significantly（P0.05） at 48 h post-transfection detected by the fluorescent observation,Real-time PCR,the immuno-histochemical analysis and RIA detection.Among which,sh2（SS 433-451） showed the highest silencing efficiency（SS mRNA reduced 59.3%,P0.05 and SS protein reduced 55.6%,P0.05 when the transfection efficiency was 68.9%）.Our result laid a basis for the animal growth promotion via the down-regulation of SS.

关 键 词：生长抑素 慢病毒载体 SHRNA 

分 类 号：S813.24[农业科学—畜牧学] Q575[农业科学—畜牧兽医]