猪繁殖与呼吸综合征病毒5'非编码区前导转录调控序列及其侧翼序列的结构与功能解析  

Structural and functional analysis for leader transcription regulating sequence and its flanking sequences in 5′ untranslated region of porcine reproductive and respiratory syndrome virus

作　　者：陆嘉琦[1,2] 高飞[1,2] 刘萍[1,2] 蔺涛[1,2] 袁世山[1,2] 

机构地区：[1]中国农业科学院上海兽医研究所 [2]中国动物卫生与流行病学中心上海分中心猪传染病研究室农业部动物寄生虫病重点实验室,上海200241

出　　处：《畜牧与兽医》2011年第11期1-6,共6页Animal Husbandry & Veterinary Medicine

基　　金：国家自然科学基金面上项目(30972204);国家重点基础研究发展计划"973计划"(2005CB523202);上海市自然科学基金项目(08ZR1422800)

摘　　要：为解析猪繁殖与呼吸综合征病毒(PRRSV)5'非翻译区(5'UTR)的高级结构与功能,以北美株PRRSV感染性克隆为平台,通过定点突变PCR技术将其5'UTR中一特殊高级调控元件的一级序列进行突变,以改变其二级茎环结构,从而解析该结构的基因调控水平。将突变DNA克隆转染入BHK-21细胞后利用免疫荧光及RT-PCR试验来研究拯救后突变病毒的转录、翻译特性,及通过空斑形态学及病毒生长曲线分析突变病毒的生长特性。结果表明,PRRSV 5'UTR中该高级调控元件的顶端环结构为病毒复制所必需,其只可耐受2个核苷酸的突变;同时发现该调控元件中一保守的茎结构为病毒复制非必需。由此证实了PRRSV 5'UTR中调控病毒复制过程的必需高级结构,为进一步解析PRRSV复制调控元件奠定基础。It is known that the 5＇ untranslated region （5＇UTR） plays a crucial role in the life cycle of positive-strand RNA viruses. In this paper, based on the porcine reproductive and respiratory syndrome virus （PRRSV） full-length infectious cDNA clone pAPRRS, a series of mutants which could modify a conserved hairpin structure were obtained through PCR-based mutagenesis, in order to analyse the secondary structure and functions of an independent functional entity in the PRRSV 5＇UTR. Then we transcribed the mutants DNA into BHK-21 cells and investigated the subgenomic transcription and protein translation in the rescued viruses by the indirect immunofluorescence assay （IFA） and RT-PCR. On the other hand, we also detected the growth feature by plaque morphology and growth curve to compare the rescued viruses with the parental virus. The results showed that the secondary structure of the conserved hairpin structure in PRRSV 5＇UTR was essential for virus replication, and that the loop of the structure could onty tolerate two nucleotides （nt） mutation. Meanwhile, it is found that the conserved stem of the hairpin structure was not necessary for virus rescue. All results demonstrated that an independent functional entity was found in PRRSV 5＇UTR.

关 键 词：猪繁殖与呼吸综合征病毒 5’非编码区 前导转录调控序列 病毒复制 基因表达调控 

分 类 号：S852.65[农业科学—基础兽医学]