高致病性猪繁殖与呼吸综合征病毒弱毒疫苗株作为外源基因表达活病毒载体的研究  

Attenuated Porcine Reproductive and Respiratory Syndrome Virus Vaccine Strain as a Live Virus Vector for Exogenous Gene Expression

作　　者：陈金霞 张宽 张玉娇 杜楠楠 郑海红[1] 李丽薇[1,2] 周艳君 虞凌雪[1,2] 童武 郑浩[1,2] 童光志 高飞[1,2] CHEN Jinxia;ZHANG Kuan;ZHANG Yujiao;DU Nannan;ZHENG Haihong;LI Liwei;ZHOU Yanjun;YU Lingxue;TONG Wu;ZHENG Hao;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3期12-19,共8页Chinese Journal of Animal Infectious Diseases

基　　金：广东省重点领域研发计划资助(2019B020211003);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：利用反向遗传操作技术,猪繁殖与呼吸综合征病毒(PRRSV)也可以被用作外源基因表达的活载体。前期研究将猪瘟病毒E2蛋白的编码基因插入了高致病性PRRSV弱毒疫苗株HuN4-F112的非结构蛋白和结构蛋白编码间区,所拯救的重组病毒rPRRSV-E2能够在体外和体内高效表达E2蛋白。能够为猪瘟和HP-PRRS的感染提供100%的免疫保护。本研究利用rPRRSV-E2的全长感染性克隆平台,在PRRSV基因组ORF7和3'非翻译区之间插入了转录调控序列6(TRS6)和绿色荧光蛋白的编码基因,构建并拯救了重组病毒rPRRSV-E2-EGFP,该病毒感染MARC-145细胞后,可以同时表达猪瘟E2蛋白和EGFP。外源序列在载体中能够保持遗传稳定。本研究为开发PRRSV作为病毒活载体疫苗提供了一个新的外源基因插入位点和构建策略,对今后多价疫苗的研发有重要意义。Porcine reproductive and respiratory syndrome virus(PRRSV)can be a living vector for exogenous gene expression by using reverse genetic manipulation technology.In the previous studies,the E2 gene of Classical swine fever virus(CSFV)was inserted into the coding region between nonstructural and structural proteins of highly pathogenic PRRSV attenuated vaccine strain HuN4-F112,and the rescued recombinant virus rPRRSV-E2 expressed E2 protein efficiently in vitro and in vivo.The recombinant rPRRSV-E2 virus provided 100%immunoprotection for the infection of CSFV and HP-PRRSV.In this study,the full-length infectious cDNA clone of rPRRSV-E2 was used as a platform and the coding gene of transcriptional regulatory sequence 6(TRS6)and green fluorescent protein were inserted between ORF7 and 3'untranslated region of PRRSV genome.The resulting recombinant virus rPRRSV-E2-EGFP was constructed and rescued,which expressed CSFV E2 protein and EGFP simultaneously when it infected MARC-145 cells.Exogenous sequences were genetically stable in the vector.In conclusion,this study provided a new foreign gene insertion site and construction strategy for the development of PRRSV as a live viral vector vaccine,which was of great significance for the future research and development of multivalent vaccines.

关 键 词：猪繁殖与呼吸综合征病毒 3'非翻译区 转录调控序列 外源基因表达 

分 类 号：S852.65[农业科学—基础兽医学]