猪生长抑素Ⅱ型受体基因(SSTR2)shRNA的构建与筛选  

Construction and Selection of shRNA against Somatostatin ReceptorⅡ Gene (SSTR2) in Swine (Sus scrofa)

作　　者：杨林[1,2] 刘小龙[1] 习欠云[1] 江青艳[1] 徐鹏如[1] 张永亮[1] 

机构地区：[1]广东省农业动物基因组学与分子育种重点实验室,华南农业大学动物科学学院,广州510642 [2]广东省医学实验动物中心,佛山528248

出　　处：《农业生物技术学报》2012年第4期382-388,共7页Journal of Agricultural Biotechnology

基　　金：广东省科技攻关项目(No.2010B020301006);广东省粤港招标项目(No.2008)

摘　　要：生长激素(GH)是调控动物生长的重要激素,GH分泌水平的高低主要受正性调控因子生长激素释放因子(GRF)和负性调控因子生长抑素(SS)的双向调节。SS通过生长抑素受体(somatostatin receptor,SSTR)发挥作用,减少GH的分泌水平。本研究设计了3条SSTR2的短发夹RNA(shRNA),构建猪(Sus scrofa)SSTR2真核表达质粒(pcDNA3.1(-)-SSTR2)和SSTR2慢病毒RNA干扰质粒(pshRNA-copGFP lentivector,LV-shRNA),并共转染中国仓鼠(Cricetulus griseus)卵巢细胞系(CHO),通过对转染细胞的荧光观察、Real-time PCR和酶联免疫法(ELISA)检测,结果表明,CHO转染细胞中猪SSTR2 mRNA表达水平不同程度的抑制,分别为90.4%、28.3%和86.3%,(P<0.05)。其中,LV-shRNA1表现出最高的沉默效率:转染效率在80%左右时,猪SSTR2mRNA水平沉默效率为90.4%,蛋白质水平降低了33.3%。本研究成功地筛选到了降低猪SSTR2基因表达的shRNA,为利用shRNA技术下调动物基因表达,构建转基因模型提供思路。Growth hormone （GH） is the main hormone which can control the growth of animals. GH release is controled by growth hormone releasing factor（GRF） and somatostatin（SS）. SS is one of important negative factors regulating GH release, reducing somatostatin receptor U （SSTR2） expression thereby decreased the inhibition of SS. In this study, According to pig （Sus scrofa） SSTR2 gene deposited in GenBank, three short hairpin RNAs （shRNA） were designed and synthesized, after annealing in vitro, the three shRNA were cloned into the pshRNA-copGFP lentivector （LV-shRNA）. Then LV-shRNA and pcDNA3.1 （-）-SSTR2 recombinant plasmid were established, transient-transfection into Chinese hamster（Cricetulus griseus） ovary cell （CHO）. The results showed, both SSTR2 mRNA and protein levels down-regulated significantly （P〈0.05） at 48 h post-transfection detected by the fluorescent observation, Real-time PCR, and Enzyme-linked imunosorbent assay （ELISA）. Expression of shRNAs in CHO cells reduced SSTR2 mRNA level by 90.4% 28.3% and 86.3% respectively. Among which, LV-shRNA1 showed the highest silencing efficiency, SSTR2 mRNA reduced 90.4 % （P〈0.05） and SSTR2 protein reduced 33.3% （P〈0.05） when the transfection efficiency was 80%. The LV-shRNA vector which down-regulated the pig SSTR2 gene expression was constructed successfully, and can facilitate further study on the transgenic pig.

关 键 词：RNAI SSTR2 慢病毒载体 猪 

分 类 号：S828.5[农业科学—畜牧学]