用于病毒拯救的稳定表达琥珀正交系统的细胞系构建及鉴定  

Establishment and identification of a cell line stably expressing amber orthogonal system for virus rescue

作　　者：龚青 王相伟 殷相平[1] 张杰 毛箬青[1] 孙跃峰 GONG Qing;WANG Xiang-wei;YIN Xiang-ping;ZHANG Jie;MAO Ruo-qing;SUN Yue-feng(State Key Laboratory of Veterinary Etiological Biology/Lanzhou Veterinary Research Institute,Chinese Academy of Agricultural Sciences,Lanzhou 730046,China;Hebei Key Laboratory of Preventive Veterinary Medicine/College of Animal Science and Technology,Hebei Normal University of Science&Technology,Qinhuangdao 066004,China)

机构地区：[1]中国农业科学院兰州兽医研究所家畜疫病病原生物学国家重点实验室,甘肃兰州730046 [2]河北科技师范学院动物科技学院河北省预防兽医学重点实验室,河北秦皇岛066004

出　　处：《中国兽医科学》2022年第4期429-438,共10页Chinese Veterinary Science

基　　金：甘肃省重点研发计划项目(20YF3NA005,21YF5WA153)。

摘　　要：复制缺陷型病毒不能在宿主体内复制,用其制备的疫苗不仅能够诱导宿主体液免疫和细胞免疫应答,还具有较好的安全性。本研究旨在构建一种利用琥珀正交系统制备复制缺陷型病毒的新平台。首先构建携带琥珀终止密码子(TAG)和EGFP报告基因的质粒(pTAG-EGFP),然后以质粒pAcBac1.tR4-MbPyl为模板,利用PCR方法扩增正交氨酰tRNA合成酶/tRNA片段(aaRS/tRNA)并克隆至不同载体中,成功构建了真核表达质粒paaRS/tRNA。将不同组合的质粒paaRS/tRNA和pTAG-EGFP共转染BHK-21细胞,荧光检测表明氨酰tRNA合成酶及tRNA在BHK-21细胞中具有正交性和高度特异性;经G418和嘌呤霉素筛选及Western-blot验证,本研究成功构建了一种稳定表达氨酰tRNA合成酶及tRNA的BHK-21细胞系,为后续拯救TAG突变复制缺陷型病毒提供了强有力的技术平台,也为重大动物疫病复制缺陷型病毒活疫苗研究奠定了基础。Vaccines generated by replication-defective viruses can not only induce humoral and cellular immune responses,but also have better safety.The aim of this study is to construct a new platform for generation of replication-defective viruses using amber orthogonal system.Firstly,plasmid(pTAG-EGFP)carrying EGFP reporter gene with an amber termination codon(TAG)was constructed.Then,the orthogonal aminoacyl tRNA synthetase/tRNA fragment(aa RS/tRNA)was amplified by PCR from p Ac Bac1.tR4-MbPyl plasmid and cloned into different vectors,and the paa RS/tRNA eukaryotic expression plasmids were successfully constructed.Different combinations of plasmids expressing aaRS/tRNA and pTAG-GFP were co-transfected into BHK-21 cells.Fluorescence detection and Western-blot results showed that the aminoacyl tRNA synthetase has orthogonality and high specificity in BHK-21 cells.Cell lines stably expressing orthogonal aminoacyl tRNA synthase and tRNA were successfully established by G418 and puromycin screening.In conclusion,this study provided a powerful platform for the subsequent rescue of replication-deficient viruses containing TAG mutation,and also laid a foundation for future study of live vaccine of replication-deficient viruses in major animal diseases.

关 键 词：非天然氨基酸 正交系统 正交氨酰tRNA合成酶/tRNA 报告基因 

分 类 号：S852.65[农业科学—基础兽医学]