猪繁殖与呼吸综合征病毒转录调控序列顺式作用元件的鉴定及其在基因转录表达中的机制解析  

Dissection for the Cis-acting Element and the Mechanism of Transcriptional Regulatory Sequence Functions in Transcription and Gene Expression of RRRSV

作　　者：张宽 陈金霞 张玉娇 杜楠楠 郑浩[1,2] 姜一峰 李丽薇[1,2] 虞凌雪 周艳君[1,2] 童武 童光志[1,2] 高飞 ZHANG Kuan;CHEN Jinxia;ZHANG Yujiao;DU Nannan;ZHENG Hao;JIANG Yifeng;LI Liwei;YU Lingxue;ZHOU Yanjun;TONG Wu;TONG Guangzhi;GAO Fei(Shanghai Veterinary Research Institute,CAAS,Shanghai 200241,China;Jiangsu Co-Innovation Center for the Prevention and Control of Important Animal Infectious Disease and Zoonoses,Yangzhou University,Yangzhou 225009,China)

机构地区：[1]中国农业科学院上海兽医研究所,上海200241 [2]扬州大学江苏省动物重要疫病与人兽共患病防控协同创新中心,扬州225009

出　　处：《中国动物传染病学报》2023年第2期13-21,共9页Chinese Journal of Animal Infectious Diseases

基　　金：国家重点研发计划政府间国际科技创新合作重点专项(2016YFE0112500);国家自然科学基金(31670158)。

摘　　要：猪繁殖与呼吸障碍综合征病毒(PRRSV)的转录调控序列(TRS)在sg mRNA的合成中起着至关重要的作用。为了深入了解TRS在病毒亚基因组转录和翻译过程中的作用,本研究通过对高致病性PRRSV细胞传代致弱株HuN4-F112的转录调控序列进行鉴定,并构建了一系列针对TRS6突变以及将TRS6替换为其他Body TRS的全长感染性克隆。结果表明,同时突变TRS6核心寡核苷酸3'端两个碱基或同时缺失5'端和3'端侧翼序列的全长突变体克隆均不能拯救出病毒,而单独缺失TRS65'端或3'端侧翼序列的全长突变体克隆可以拯救出病毒,但侧翼序列缺失后拯救出的病毒相较于原始亲本病毒的滴度有所下降;将HuN4-F112-EGFP的TRS6替换为TRS2、3、7均可以拯救出病毒,并有相似的生长特性,但含有TRS2的重组病毒滴度相对较低,而将TRS6替换为TRS4或TRS5后,无法拯救出病毒。本研究鉴定了HuN4-F112的一系列Body TRS在基因组中的相对位置,同时也证明了ORF1b和ORF2a基因区中4种不同的Body TRS都可以引导基因稳定高效的表达,该研究为今后以PRRSV疫苗株为活病毒载体进行多价重组疫苗的研发奠定了基础。The transcription regulatory sequence(TRS)of the Porcine reproductive and respiratory syndrome virus(PRRSV)plays an important role in the synthesis of sg mRNA.In order to deeply understand the role of TRS in viral sg mRNA transcription and translation,we first identified the transcription regulatory sequences of PRRSV strain HuN4-F112.A series of mutant full-length infectious clones with extra inserted TRS6 based on the full-length infectious cloning platform of HuN4-F112-EGFP expressing green fl uorescent protein(EGFP)were constructed,and the full-length infectious clones with other Body TRS inserted were also constructed.Through the study of virus rescue,the full-length mutant clone with two bases at the 3'of the core oligonucleotide of TRS6 couldn’t be rescued,but the full-length mutant clone with 5'and 3'fl anking sequences deleted alone could be rescued,while the full-length mutant clone with 5'and 3'fl anking sequences both deleted couldn’t be rescued,but the titer of the virus with deletion of fl anking sequences is lower than that of the original parent virus.In HuN4-F112-EGFP,TRS6 was replaced by TRS2,3 and 7.The live virus could be rescued and have similar growth characteristics,but the titer of recombinant virus containing TRS2 is relatively low.However,after TRS6 was replaced by TRS4 and TRS5,the virus couldn’t be rescued.In this study,the relative position of a series of TRS-Bodies of HuN4-F112 in genome was identified.In addition,it was proved that 4 different Body TRS in ORF1b and ORF2a gene regions could guide the stable and efficient expression of genes,which laid a foundation for the research and development of multivalent recombinant vaccines using PRRSV vaccine strains as live virus vectors in the future.

关 键 词：猪繁殖与呼吸综合征病毒 转录调控序列 非连续性转录 

分 类 号：S852.65[农业科学—基础兽医学]