H5N1型流感病毒灭活疫苗(MDCK细胞)原液蛋白成分分析  

Analysis of protein composition of H5N1 influenza virus inactivated vaccine(MDCK cells) bulks

作　　者：刘博 张青梅 邱冉 李芳 彭飞霞 张文佳 苏敏讷 张哲罡 张家友 杨晓明 LIU Bo;ZHANG Qingmei;QIU Ran;LI Fang;PENG Feixia;ZHANG Wenjia;SU Minne;ZHANG Zhegang;ZHANG Jiayou;YANG Xiaoming(Wuhan Institute of Biological Products Co.,Ltd.,Wuhan 430207,Hubei Province,China;不详)

机构地区：[1]武汉生物制品研究所有限责任公司,湖北武汉430207 [2]中国生物技术股份有限公司,北京100024

出　　处：《中国生物制品学杂志》2023年第4期385-388,394,共5页Chinese Journal of Biologicals

基　　金：湖北省重点研发计划(2020DCC002)。

摘　　要：目的 对H5N1型流感病毒灭活疫苗(MDCK细胞)原液蛋白成分进行分析,为完善该疫苗的质量控制方法及疫苗的研发提供依据。方法 将H5N1型流感病毒株接种于MDCK细胞,病毒培养48 h后,收获病毒液,经澄清、超滤浓缩、β-丙内酯灭活、Capto Q和Sephorase 4FF两步层析纯化,获得原液样品。应用透射电镜分析样品中病毒颗粒的形态;单向免疫扩散(single radial immunodiffusion,SRID)试验鉴别病毒原液血凝素(hemagglutinin,HA);高效液相色谱进行纯度分析;梯度SDS-PAGE进行蛋白电泳分析;液质联用仪(LC/MS)结合对回收的各条带蛋白多肽进行二级质谱序列测定的方法分析样品中所含的蛋白成分。结果 在透射电镜下可观察到80~120 nm球形H5N1流感病毒颗粒,呈典型的流感病毒形态;鉴别试验表明病毒原液抗原性与病毒株相一致;经梯度SDS-PAGE分析,病毒原液有2条带;LC/MS质谱分析显示,原液样品中H5N1型流感病毒蛋白为主要成分,包括H5N1型流感病毒的NP、M1、HA、NA等蛋白。结论 对H5N1型流感病毒灭活疫苗制备过程中原液的蛋白成分进行了分析,为该类疫苗的研发及质量控制提供了参考依据。Objective To analyze the protein components in the bulks of H5N1 inactivated influenza virus vaccine(MDCK cells),providing experimental basis for the improvement of the quality control method and the development of the vaccine.MethodsThe H5N1 influenza virus strain was inoculated into MDCK cells.After culturing the virus for 48 h,the virus liquid was harvested,and the original liquid sample was obtained by clarification and ultrafiltration concentration,β-propiolactone inactivation,and two-step chromatography purification with Capto Q and Sephorase 4FF.Morphology of virus particles in the sample was analyzed by transmission electron microscopy,while hemagglutinin identification of the virus bulk by single radial immunodiffusion(SRID)assay,purity by high performance liquid chromatography,and protein electrophoresis by gradient SDS-PAGE.The protein components contained in the samples were analyzed by liquid chromatography-mass spectrometry(LC/MS)combined with secondary mass spectrometry sequence determination of the recovered protein polypeptides.ResultsSpherical H5N1 influenza virus particles of 80 ~ 120 nm were observed under transmission electron microscope,showing the typical shape of influenza virus.The identification test showed that the antigenicity of the virus bulks was consistent with the virus strain.The gradient SDS-PAGE analysis showed that the virus bulk had two bands.LC/MS mass spectrometry analysis showed that H5N1 influenza virus protein was the main component in the bulk samples,including NP,M1,HA,NA and other proteins of H5N1 influenza virus.ConclusionThe protein composition of the bulk during the preparation of H5N1 influenza virus inactivated vaccine was analyzed,which provided a reference for the development and quality control of this type of vaccine.

关 键 词：H5N1型流感病毒 疫苗 病毒原液 质谱分析 

分 类 号：R181.1[医药卫生—流行病学]