miR-155体外特异性靶向鸡内源性反转录病毒ALVE1 env转录物的研究  

Studies on avian endogenous retrovirus ALVE1 env transcripts specifically targeted by miR-155 in vitro

作　　者：朱文奇[1,2] 胡序明[1,2] 陈世豪[1,2] 刘洋洋[1,2] 孙振[1,2] 耿拓宇[1,2] 宋成义[1,2] 高波[1,2] 崔恒宓[1,2] 

机构地区：[1]扬州大学表观遗传学及表观基因组学研究所,江苏扬州225009 [2]扬州大学动物科学技术学院,江苏扬州225009

出　　处：《畜牧与兽医》2016年第5期7-11,共5页Animal Husbandry & Veterinary Medicine

基　　金：国家973项目课题(2012CB517605);国家自然科学基金(81171965;81372237;91540117);江苏省畜牧学优势学科项目;中国博士后科学基金第57批面上资助项目(2015M571828)

摘　　要：通过生物信息学方法,发现了鸡内源性反转录病毒禽白血病E1(ALVE1)的env转录物存在gga-miR-155作用位点(AGCATTA),并在体外验证其靶位点的活性。将鸡胚成纤维细胞(CEF)基因组中扩增的ALVE1 env转录物构建至荧光素酶报告载体pmir GLO,获得重组质粒pmirGLO-ALVE1-ENV-WT(野生型),并利用重叠PCR将其miR-155作用位点AGCATTA突变为ATTCAAA,然后构建重组质粒pmir GLO-ALVE1-ENV-MU(突变型),同时将gga-miR-155前体序列构建至pc DNA3.1载体,获得重组质粒pc DNA3.1-gga-miR-155,将这些质粒共转染至DF-1细胞中,48 h后收集细胞并检测荧光素酶活性。结果发现:野生组能显著下调pmir GLO-ALVE1-ENV-WT荧光素酶活性,而对照组无显著改变;对照组和突变组均未能改变pmir GLO-ALVE1-ENV-MU荧光素酶活性。本研究证实gga-miR-155可直接靶向ALVE1 env转录物,为鸡内源性反转录病毒的调控机制提供了新的启示。In this study,the DNA gga-miR-155 target site AGCATTA was demonstrated to exist in env transcripts of endogenous retrovirus ALVE1 by bioinformatics analysis. ALVE1 env sequence was amplified from chicken embryo fibroblast（ CEF） cell genome and subcloned into pmirG LO vector which contains luciferase gene,thus creating the recombinant plasmid＂pmirG LO-ALVE1-ENV-WT＂. Meanwhile,overlap PCR was performed to obtain the mutate sequences of miR-155 target site and the recombinant plasmid＂pmirG LO-ALVE1-ENV-MU＂was constructed. In addition,gga-miR-155 precursor sequence was cloned into pcD NA 3. 1 vector,and the recombinant plasmid＂pcD NAgga-miR-155＂was created. Then,these vectors were co-transfected into DF-1 cells and the cells were collected to detect their luciferase activity. The result indicated that gga-miR-155 significantly reduced luciferase activity of pmirG LO-ALVE1-ENV-WT,while in all of control groups and the mutant group no significant change was found in luciferase activity. The results of fluorescence quantitative PCR further revealed that gga-miR-155 agomir in MSB1 cells significantly down-regulated the expression of ALVE1 env transcripts. This study suggests that gga-miR-155 may directly target ALVE1 env transcript. This is the first report that miR- 155 can directly target endogenous retroviral sequences.

关 键 词：MIR-155 靶位点 鸡内源性反转录病毒 ALVE1 ENV 

分 类 号：S852.65[农业科学—基础兽医学]