基于FMDV 2A肽的新城疫病毒共表达系统的构建及验证  

Construction and validation of a Newcastle disease virus co-expression system based on FMDV 2A peptide

作　　者：王海蓉 李泽典 任园园 陈建 余祖华 魏颖[1,2] 丁轲 陈松彪 贾艳艳[1,2] 郁川 张春杰 何雷[1,2] WANG Hai-rong;LI Ze-dian;REN Yuan-yuan;CHEN Jian;YU Zu-hua;WEI Ying;DING Ke;CHEN Song-biao;JIA Yan-yan;YU Chuan;ZHANG Chun-jie;HE Lei(Luoyang Key Laboratory of Live Carrier Biomaterial and A nimal Disease Prevention and Control,Luoyang 471003,China;Laboratory of Functional Microbiology and Animal Health,Henan University of Science and Technology,College of Animal Science and Technology,Henan University of Science and Technology,Luoyang 471003,China;Luoyang Vocational and Technical College,Luoyang 471003,China)

机构地区：[1]洛阳市活载体生物材料与动物疫病防控重点实验室,河南洛阳471003 [2]河南科技大学功能微生物与畜禽健康实验室,河南科技大学动物科技学院,河南洛阳471003 [3]洛阳职业技术学院,河南洛阳471003

出　　处：《中国兽医科学》2023年第2期198-206,共9页Chinese Veterinary Science

基　　金：国家自然科学基金面上项目(32072771);河南省科技攻关计划项目(222102110012,192102110078);大学生研究训练计划(SRTP)项目(2022491)。

摘　　要：为构建表达双外源基因的重组新城疫病毒(NDV)反向遗传学表达体系,本研究利用口蹄疫病毒(FMDV)2A序列构建同时表达增强型绿色荧光蛋白(EGFP)基因和红色荧光蛋白(RFP)基因的重组NDV(rLS-EGFP-F2A-RFP),并检测双外源蛋白的表达水平。生物学特性检测结果显示,重组病毒保持了亲本La Sota株的鸡胚高滴度生长以及低致病力的遗传特性,生长曲线分析表明,重组病毒具有与亲本毒株相似的生长动力学特性,在细胞培养中能够稳定复制;荧光观察结果表明,EGFP和RFP蛋白在重组病毒感染细胞中均获得表达且具有生物学活性;Western-blot试验结果表明,F2A肽在重组NDV表达系统中发挥了良好的自剪切功能,双外源蛋白均可独立表达。本研究为NDV多联疫苗的研发提供实验依据和技术平台。To construct the co-expression of double foreign genes system of recombinant Newcastle disease virus(r NDV),the r NDV(r LS-EGFP-F2A-RFP)co-expressing the enhanced green fluorescent protein(EGFP)gene and red fluorescent protein(RFP)gene was constructed using the 2A sequence of foot-andmouth disease virus(FMDV).The expression levels of double foreign proteins were explored.The biological characteristics results showed that the r NDV maintained the excellent genetic characteristics with high growth titer and low virulence.The growth curve analysis showed that r LS-EGFP-F2A-RFP could replicate stably in host cells and had similar growth kinetics characteristics compared to its parental strain.The fluorescence observation indicated that both EGFP and RFP proteins were successfully expressed and biologically active in recombinant virus infected cells.Western-blot results show that F2A peptide played a favorable self-cleavage function in this expression system,and both foreign proteins were expressed independently.This study provides an experimental platform for the development of recombinant NDV multiplex vaccine.

关 键 词：重组新城疫病毒 F2A肽 EGFP RFP 共表达体系 

分 类 号：S852.659.5[农业科学—基础兽医学]