表达分泌荧光素酶的重组流感病毒构建及体外生物学特性研究  

Construction of recombinant influenza virus expressing Gaussia luciferase and analysis of its in vitro biological characteristics

作　　者：王东红[1] 邓瑶[1] 叶飞[1] 周剑芳[2] 王文[1] 黄保英[1] 王梦微 孟昕[1] 谭文杰[1] Wang Donghong;Deng Yao;Ye Fei;Zhou Jianfang;Wang Wen;Huang Baoying;Wang Mengwei;Meng Xin;Tan Wenjie(National Health Commission Key Laboratory of Biosafety,National Institute for Viral Disease Control and Prevention,Chinese Center for Disease Control and Prevention,Beijing 102206,China;World Health Organization Collaborating Center for Reference and Research on Influenza,Institute for Viral Disease Control and Prevention,Chinese Center for Disease Control and Prevention,Beijing 102206,China)

机构地区：[1]中国疾病预防控制中心病毒病预防控制所,国家卫生健康委员会生物安全重点实验室,北京102206 [2]中国疾病预防控制中心病毒病预防控制所,世界卫生组织流感参比和研究合作中心,北京102206

出　　处：《中华微生物学和免疫学杂志》2023年第11期823-828,共6页Chinese Journal of Microbiology and Immunology

基　　金：国家自然科学基金(82041041);国家重点研发计划(2022YFC2304100)。

摘　　要：目的采用不同的流感病毒骨架构建表达分泌荧光素酶(Gaussia luciferase,Gluc)的重组流感病毒,同时研究其生长特性、遗传稳定性、Gluc表达水平、体外抗流感药物活性。方法在PR8NA的C端插入猪捷申病毒2A肽(porcine teschovirus-2A autocleavage peptide,P2A)自剪切位点及Gluc编码基因,其余7个质粒分别来源于A/Puerto Rico/8/34(PR8)(H1N1)和A/WSN/1933(WSN)(H1N1),通过流感病毒反向遗传学8质粒系统拯救重组病毒,分别命名为PR8NAGluc/PR8和PR8NAGluc/WSN。检测重组病毒遗传稳定性;对比PR8NAGluc/PR8和PR8NAGluc/WSN的荧光强度;检测重组病毒的生长动力学;同时测定PR8NAGluc/WSN荧光强度与半数组织培养感染剂量(median tissue culture infective dose,TCID_(50))的相关性及对奥司他韦、法匹拉韦和莲花清瘟的体外抗流感药物活性。结果通过反向遗传学成功拯救出以PR8和WSN为骨架表达Gluc的重组病毒。相对于PR8骨架,以WSN为骨架明显提高了Gluc的表达且遗传稳定,复制动力学比野生型稍低。PR8NAGluc/WSN荧光强度与TCID_(50)有较好的相关性,其对奥司他韦、法匹拉韦和莲花清瘟具有良好的敏感性。结论以WSN为骨架的重组病毒荧光表达强度比PR8骨架高,为高通量筛选抗流感病毒药物及研发流感病毒载体疫苗提供参考。ObjectiveTo construct recombinant influenza viruses expressing Gaussia luciferase(Gluc)with different influenza virus backbones and analyze their growth characteristics,genetic stability,ability to express Gluc and in vitro anti-influenza drug activity.MethodsThe C-terminal of PR8NA was modified by inserting the porcine teschovirus-2A autocleavage peptide(P2A)and the Gluc-coding gene.Recombinant viruses,PR8NAGluc/PR8 and PR8NAGluc/WSN,were rescued using the eight-plasmid system of influenza virus reverse genetics,with seven plasmids derived from A/Puerto Rico/8/34(PR8)(H1N1)and A/WSN/1933(WSN)H1N1.The genetic stability of the recombinant viruses was verified by RT-PCR.The fluorescence activity and the growth kinetics of the two recombinant viruses were compared.The correlation between the fluorescence activity of PR8NAGluc/WSN and median tissue culture infective dose(TCID_(50)),and the anti-drug activity of PR8NAGluc/WSN against oseltamivir,favipiravir,and Lianhua Qingwen in vitro were also analyzed.ResultsThe Gluc-expressing recombinant viruses constructed using PR8 and WSN backbones were successfully rescued by reverse genetics.Compared with the PR8 backbone,the WSN backbone significantly improved the fluorescence activity of Gluc.Moreover,the PR8NAGluc/WSN virus expressed stably in embryonated egg,and its replication kinetics was slightly lower than that of wild type.The fluorescence activity of PR8NAGluc/WSN virus had a good correlation with its TCID_(50).The PR8NAGluc/WSN virus was sensitive to oseltamivir,favipiravir and Lianhua Qingwen.ConclusionsThe recombinant virus with a WSN backbone exhibited higher fluorescence expression intensity as compared with the recombinant virus with a PR8 backbone.This study provided reference for high-throughput screening of anti-influenza drugs and the development of influenza virus vector vaccines.

关 键 词：流感病毒 反向遗传学 荧光素酶 A/WSN/1933(H1N1) 重组病毒 

分 类 号：R373[医药卫生—病原生物学]