摘要
【目的】当前中国牛群中流行的丁型流感病毒多属D/Yama2019遗传进化谱系,构建此遗传进化谱系丁型流感病毒的反向遗传操作系统,为深入探究其复制与致病机制提供研究工具。【方法】以中国丁型流感病毒株D/bovine/CHN/JY3002/2022(简称D/JY3002,属D/Yama2019遗传进化谱系)为研究材料,将编码该病毒主要抗原血凝素-酯酶-融合蛋白(hemagglutininesterase-fusion, HEF)的基因组节段对应的DNA片段,无缝克隆至经设计改造的双向表达载体pCC1-DualPro中;其余基因组节段对应的DNA片段,则无缝克隆至常用双向表达载体pHW2000中。随后经改进优化的操作步骤,拯救出重组丁型流感病毒。【结果】拯救的丁型流感病毒株(rD/JY3002)与天然分离的中国丁型流感病毒株(D/JY3002)在复制能力上表现相当,且二者复制动力学特征相仿。rD/JY3002至少可稳定传代5代。利用已建立的反向遗传操作系统,拯救出携带绿色荧光报告基因GFP以及其他遗传进化谱系(D/OK和D/660)丁型流感病毒HEF基因的重组丁型流感病毒,分别命名为rD/JY3002-GFP、rD/JY3002-D/OK-HEF和rD/JY3002-D/660-HEF,证实了不同遗传进化谱系的丁型流感病毒间可发生基因组节段重配。【结论】建立了高效、稳定的中国丁型流感病毒株反向遗传操作系统,该系统可用于进一步开发以丁型流感病毒为载体的外源基因呈递技术。
【Objective】The current prevalent influenza D virus in Chinese cattle herds primarily belongs to the D/Yama2019 genetic lineage.The development of a reverse genetics system for the D/Yama2019 genetic lineage influenza D virus offers a crucial research tool for the in-depth investigation of its replication and pathogenic mechanisms.【Method】The Chinese influenza D virus strain D/bovine/CHN/JY3002/2022(Abbreviated as D/JY3002),belonging to the D/Yama2019 genetic lineage,was utilized as the research material.The DNA fragments corresponding to the genomic segments that encode the primary antigen of the virus,Hemagglutinin-Esterase-Fusion(HEF),were successfully cloned into the engineered bidirectional expression vector pCC1-DualPro.The DNA fragments corresponding to the remaining genomic segments were seamlessly cloned into the widely utilized bidirectional expression vector pHW2000.Subsequently,the recombinant influenza D virus was successfully rescued through improved and optimized operational procedures.【Result】The rescued influenza D virus strain(rD/JY3002)demonstrated a replication ability comparable to that of the naturally isolated Chinese influenza D virus strain(D/JY3002),and the replication kinetics of the two viruses are similar.Moreover,the rD/JY3002 virus can be stably propagated for at least five generations.Using the established reverse genetics system,recombinant influenza D viruses containing the green fluorescence reporter gene(GFP)and the HEF genes from other genetic lineages(D/OK and D/660)were successfully generated.They were respectively designated as rD/JY3002-GFP,rD/JY3002-D/OK-HEF,and rD/JY3002-D/660-HEF.These results confirmed that reassortment occurred among influenza D viruses from different genetic lineages.【Conclusion】An efficient and stable reverse genetics system for the influenza D virus Chinese strain has been established.This system can be utilized to further advance exogenous gene presentation technology,employing the influenza D virus as a vector.
作者
胡万可
陈雲霞
罗帝洲
吴斯宇
李建波
翟少伦
巨向红
廖明
魏文康
余界石
HU Wan-ke;CHEN Yun-xia;LUO Di-zhou;WU Si-yu;LI Jian-bo;ZHAI Shao-lun;JU Xiang-hong;LIAO Ming;WEI Wen-kang;YU Jie-shi(State Key Laboratory of Swine and Poultry Breeding Industry,Agro-Biological Gene Research Center,Guangdong Academy of Agricultural Sciences,Guangzhou 510640;College of Coastal Agricultural Sciences,Guangdong Ocean University,Zhanjiang 524088;College of Veteri-nary Medicine,South China Agricultural University,Guangzhou 510642;Shanwei Animal Disease Control Center,Shanwei 516601;Key Laboratory of Livestock Disease Prevention of Guangdong Province,Institute of Animal Health,Guangdong Academy of Agricultural Sciences,Guangzhou 510640;College of Animal Science and Technology,Zhongkai University of Agriculture and Engineering,Guangzhou 510225)
出处
《生物技术通报》
北大核心
2025年第12期95-105,共11页
Biotechnology Bulletin
基金
国家自然科学基金青年项目(32202795)
广东省自然科学基金面上项目(2024A1515011049)
广东省农业科学院“中青年学科带头人”“金颖之星”培养项目(R2023PY-JX025)
广东省农业科学院人才专项(青年科技骨干)项目(R2021YJ-QG008)。
