A型鸭肝炎病毒微基因组的构建  

Construction of the Mini-genome of Duck Hepatitis A Virus

作　　者：梁瑞英[1] 胡文[2] 李宁[3] 缪秋红[1] 毕庄莉 孟春春[1] 李传峰[1] 陈宗艳[1] 刘光清[1] 

机构地区：[1]中国农业科学院上海兽医研究所,上海200241 [2]甘肃农业大学,兰州730050 [3]安徽农业大学,合肥230036

出　　处：《畜牧兽医学报》2014年第4期609-613,共5页ACTA VETERINARIA ET ZOOTECHNICA SINICA

基　　金：国家自然科学基金项目(31270194;31101848;31300141);公益性农业科研专项(201003012;201303046;2014JB14);"863"计划(2011AA10A200)

摘　　要：为研究A型鸭肝炎病毒(DHAV)非编码区的结构和功能,笔者拟构建该病毒的微基因组并对其进行初步鉴定。采用酶切的方法将萤火虫荧光素酶报告基因(fLuc)与A型鸭肝炎病毒的ORF进行替换;并在5′UTR上游插入海肾荧光素酶报告基因Rluc作为内参基因;同时还在Rluc上游引入锤头状核酶,3′UTR下游引入丁型肝炎核酶,至此得到了含有双报告基因的A型鸭肝炎病毒微基因组(pRluc-fLuc)。将pRluc-fLuc转染DF-1细胞,8h便可以检测到报告基因的表达,24h表达量达到峰值。上述结果表明,已经成功构建了A型鸭肝炎病毒微基因组,这为进一步研究病毒非编码区的结构和功能以及病毒的翻译或复制的调控机理提供了良好的技术平台。In this study we constructed and identified the mini-genome of Duck Hepatitis A virus(DHAV),for analysis of the functional and structural properties of DHAV un-translated region(UTR).To construct a bicistronic of DHAV mini-genome(pRluc-fLuc),the ORF of DHAV was replaced by firefly luciferase(fLuc)gene through enzyme digestion and the Renilla luciferase(Rluc)gene was inserted before 5′UTR.We also added the hammerhead ribozyme ahead Rluc gene and the hepatitis delta virus ribozyme behind 3′UTR.We transfected the pRluc-fLuc into DF-1cells,the fLuc gene expression could be detected at 8hours post transfected(h.p.t.)and with the levels peaking was at 24h.p.t.The results showed that the DHAV mini-genome was constructed successfully and can be used for explore the role of UTR in virus transcription and translation.

关 键 词：A型鸭肝炎病毒 微基因组 非编码区 

分 类 号：S852.657[农业科学—基础兽医学]