汉坦病毒Vero细胞适应株的筛选及鉴定  

Screening and identification of Hantaan virus Vero cell adaptation strains

作　　者：管亚博 邢坤鹏 刘晨阳 王颖 杜翔宇 陈娜娜 张朔 孙宏亮 李景良 GUAN Yabo;XING Kunpeng;LIU Chenyang;WANG Ying;DU Xiangyu;CHEN Nana;ZHANG Shuo;SUN Hongliang;LI Jingliang(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》2024年第7期779-787,共9页Chinese Journal of Biologicals

基　　金：吉林省科技发展计划(20210204206YY)。

摘　　要：目的 探讨汉坦病毒(Hantataan virus,HTNV)PS-6株经乳鼠鼠脑传代后在Vero细胞上的适应性,并筛选出适应Vero细胞的高滴度HTNV。方法 将HTNV PS-6株在乳鼠鼠脑内连续传代培养后,Vero细胞上连续传10代,观察细胞病变效应(cytopathic effect,CPE),并采用蚀斑法检测病毒感染性滴度,滴度升高后,采用蚀斑克隆纯化筛选单克隆病毒并检测病毒滴度;分别以0.01、0.05、0.1 MOI感染Vero细胞后绘制病毒生长曲线;Reed-Muench法计算病毒LD50;Western blot法鉴定病毒特异性;透射电镜观察病毒大小及结构;与HTNV PS-6株全基因组核苷酸序列及氨基酸序列进行比较分析。将筛选出的Vero细胞适应株V-PS-6经腹腔注射10只BALB/c雌性小鼠,濒死时解剖,取心、肝、脾、肾、脑、肺、小肠、肌肉等组织进行免疫组化分析。结果 鼠脑传代毒株对Vero细胞具有较好适应性,初始病毒滴度为1.3 lgPFU/mL,随着传代次数增加,病毒滴度逐渐升高,稳定在7.5~7.9 lgPFU/mL;筛选出的适应株V-PS-6蚀斑边界清晰、圆润、肉眼可见,大小如针尖,LD_(50)为10-^(7.8),在相对分子质量约50 000处可见特异性蛋白条带,大小与适应前HTNV PS-6株相符;V-PS-6病毒直径为80~210 nm,与HTNV PS-6电镜结构未见明显差异,核苷酸序列同源性为99.39%,氨基酸序列同源性为98.85%;V-PS-6病毒明显分布在小鼠肾脏、肝脏、小肠、后腿肌肉及心脏中。结论 成功筛选出1株可较好适应Vero细胞的高滴度HTNV株,且具有良好的遗传稳定性,为后续HTNV Vero细胞疫苗的研发及相关机制的研究奠定了基础。Objective To study the adaptability of Hantaan virus(HTNV) PS-6 strain in Vero cells after passing through the brain of suckling mice,and to screen out a high titer HTNV strain adaptable to Vero cells.Methods HTNV PS-6 strain was cultured continuously in the brain of suckling mice,and then continued to pass on Vero cells for 10 times.The cytopathic effect(CPE) was observed for each generation of the virus,and the virus titer was determined by plaque method.After the virus titer increased on Vero cells,the monoclonal virus was screened by plaque cloning and purification and the titer of monoclonal virus was measured.After infecting Vero cells at MOIs of 0.01,0.05,and 0.1,the growth curve of the virus was drawn.The LD_(50) of the virus was calculated by Reed-Muench method,the specificity of the virus was initially identified by Western blot,and the size and structure of the virus before and after screening were observed by transmission electron microscopy.The nucleotide sequences of the whole genome and amino acid sequences were compared with those of HTNV PS-6 strain.The strain adaptive to Vero cells,V-PS-6,was injected intraperitoneally into 10 female BALB/c mice,which were dissected when dying.The tissues of heart,liver,spleen,kidney,brain,lung,small intestine and muscle were taken and analyzed by immunohistochemistry.Results The passage strain in mouse brain had good adaptability to Vero cells,with an initial virus titer of 1.3 lgPFU/mL,and the virus titer gradually increased with the increase of passage times and stabilized at 7.5-7.9 lgPFU/mL.The plaque boundary of the adapted strain V-PS-6 was clear,round and visible to naked eyes,the size was like a needle tip,the LD_(50) was 10~(-7.8),and the specific protein band was found at the relative molecular mass of about 50 000,consistent with that of the pre-adapted virus strain HTNV PS-6.The diameter of V-PS-6 strain was80-210 nm,with no significant difference in structure between V-PS-6 strain and HTNV PS-6 strain under electron microscope,and the homology of n

关 键 词：汉坦病毒 VERO细胞 适应性传代 蚀斑法 病毒滴度 

分 类 号：R373.9[医药卫生—病原生物学]