适应悬浮MDCK细胞A型流感病毒株的筛选及拯救  

Screening and rescue of influenza A virus strain adapted to suspension MDCK cells

作　　者：相振环 李臣锋 刘莹 隋丽娜 王宇田 孙艳军 吴业红 XIANG Zhenhuan;LI Chenfeng;LIU Ying;SUI Lina;WANG Yutian;SUN Yanjun;WU Yehong(Changchun Institute of Biological Products Co.,Ltd.,Changchun 130012,Jilin Province,China)

机构地区：[1]长春生物制品研究所有限责任公司,吉林长春130012

出　　处：《中国生物制品学杂志》2025年第1期1-7,共7页Chinese Journal of Biologicals

基　　金：吉林省科技发展计划项目(20210204082YY)。

摘　　要：目的筛选并拯救适应在悬浮MDCK细胞中增殖的A型流感病毒株,以期为细胞流感疫苗株的制备及筛选提供实验依据。方法将A/Nebraska/14/2019(H1N1)及A/Guangdong-Maonan/SWL1536/2019_CNIC-1909(H1N1)2株流感毒株分别于悬浮MDCK细胞中进行连续适应性传代,通过检测血凝滴度及细胞培养半数感染量(CCID50)筛选稳定适应悬浮MDCK细胞培养的毒株,并以其为亲本株,采用反向遗传学技术拯救获得流感病毒骨架毒株reH1N1及重组毒株rcH1N1,电镜下观察病毒粒子形态,经空斑纯化后进行序列验证。将纯化重组毒株rcH1N1在悬浮MDCK细胞中进行连续传代培养,检测血凝滴度及CCID50,并验证其遗传稳定性。结果毒株A/Guangdong-Maonan/SWL1536/2019_CNIC-1909的血凝滴度和CCID50更稳定,更适应悬浮MDCK细胞培养。拯救获得的重组毒株rcH1N1及骨架毒株reH1N1的病毒粒子形态均具有流感病毒粒子形态特征,空斑形成单位分别为8×10^(7)及6×10^(7)PFU,各段基因的PCR产物序列与重组质粒序列一致。重组毒株rcH1N1经5次传代后可获得较高滴度且稳定的毒株,血凝滴度最高为1∶512,lgCCID50最高为7.57,且传代毒株遗传物质保持稳定。结论筛选及拯救获得了适应悬浮MDCK细胞的A型流感病毒株rcH1N1,为细胞流感疫苗的研发奠定了基础。Objective To screen and rescue an influenza A virus strain adapted to suspension MDCK cells in order to provide an experimental basis for the preparation and screening of cell-based influenza vaccine strains.Methods Two influenza virus strains,A/Nebraska/14/2019(H1N1)and A/Guangdong-Maonan/SWL1536/2019_CNIC-1909(H1N1),were subcultured in suspension MDCK cells for continuous adaptive passaging.One strain stably adapted to suspension MDCK cell culture was screened by detecting hemagglutination titer and cell culture infective dose 50%(CCID_(50)).This strain was used as the parental virus for subsequent reverse genetics manipulation to obtain the influenza backbone strain reH1N1 and reassor-tant strain rcH1N1.The virus particle morphology was observed under electron microscope,and the sequence was verified by plaque purification.The purified recombinant strain rcH1N1 was continuously passaged in suspension MDCK cells,the hemagglutination titer and CCID_(50)were detected,and its genetic stability was verified.Results The hemagglutination titer and CCID_(50)of strain A/Guangdong-Maonan/SWL1536/2019_CNIC-1909 were more stable and more adapted to suspension MDCK cell culture.The rescued reassortant strain rcH1N1 and the backbone strain reH1N1 had the morphological characteristics of influenza virus particles.The plaque forming units were 8×10^(7)and 6×10^(7)PFU,respectively,and the PCR product sequences of each gene segment were consistent with those of the recombinant plasmid.The reassortant strain rcH1N1 obtained stable strain with high titer after five passages,the hemagglutination titer reached a maximum of 1∶512,with an lgCCID_(50)of up to 7.57,and the genetic material of the passage strain remained stable.Conclusion The influenza A virus strain rcH1N1 adapted to suspension MDCK cells was screened and rescued,which lays a foundation for the development of cell-based influenza vaccines.

关 键 词：A型流感病毒株 反向遗传学 悬浮培养 MDCK细胞 细胞流感疫苗 

分 类 号：R373.31[医药卫生—病原生物学]