适用于生产H5N1流感疫苗的MDCK亚克隆细胞的筛选及检定  

Cloning and analyzing of MDCK cells for H5N1 influenza vaccine production

作　　者：黄晓媛 张家友 刘洋 赵巍 施金荣 乐欣如 喻刚 韩锡鑫 杨晓明 Huang Xiaoyuan;Zhang Jiayou;Liu Yang;Zhao Wei;Shi Jinrong;Le Xinru;Yu Gang;Han Xixin;Yang Xiaoming(The Influenza Group of Wuhan Institute of Biological Products Co.Ltd.,Wuhan 430207,China;National Engineering Technology Research Center of Combined Vaccine,Wuhan 430207,China;China National Biotec Group Co.Ltd.,Beijing 100029,China)

机构地区：[1]武汉生物制品研究所有限责任公司流感疫苗课题组,430207 [2]国家联合疫苗工程技术研究中心,武汉430207 [3]中国生物技术股份有限公司,北京100029

出　　处：《中华微生物学和免疫学杂志》2019年第2期81-87,共7页Chinese Journal of Microbiology and Immunology

摘　　要：目的筛选对H5N1流感病毒高度敏感、低致瘤性的MDCK细胞亚株,并对该细胞株进行全面的检定及分析。方法通过极限稀释法获得细胞亚株,再对各细胞亚株进行血凝滴度、受体丰度和致瘤性检测,筛选出安全性好、敏感性高的细胞亚株,然后对所筛选细胞亚株进行全面的检定。结果通过细胞克隆试验,共得到108株单细胞亚克隆株,经两轮血凝滴度初筛后得到3株细胞,再经致瘤性及受体丰度比较后选定一株亚克隆株G1作为备选细胞株。核型分析显示该亚细胞株染色体数目众数为78,未发现细菌、真菌、支原体及外源因子污染。裸鼠细胞致瘤性试验显示在观察期末接种部位病理解剖与阴性对照组无显著性差异,细胞未显示明显致瘤性。以细胞裂解物和细胞DNA接种裸鼠,均未致裸鼠产生结节,病理解剖显示与对照组无显著性差异,细胞未显示致癌性。结论筛选出的MDCK-G1亚细胞株符合安全性和敏感性的要求,具有用于大流行流感疫苗生产的潜在可能性。To screen a Madin-Darby canine kidney(MDCK)cell line for H5N1 influenza virus isolation and to evaluate its safety in vaccine production.Methods MDCK cells were cloned by the method of limiting dilution.Hemagglutination test was used to screen MDCK cells that were suitable for H5N1 influenza virus production.Tests for analyzing the characteristics,extraneous agents,endogenous agents and tumorigenicity of MDCK cells were performed according to Chinese Pharmacopeia Volume Ⅲ.Results A total of 108 MDCK cell lines were obtained and three of them were selected after hemagglutination test.G1 cells were chosen following further screening with tumorigenicity test and receptor abundance analysis.The average number of chromosomes of the MDCK-G1 cells was 78±4.No bacteria,fungi or mycoplasma contamination was detected.In experimental group,each nude mouse was injected with 1×107/ml viable cells to observe their tumorigenicity.Twelve weeks after cell injection,no node was found at injection sites or in gross anatomy.There was no significant difference between the experimental and negative control groups.The result of the tumorigenicity test was negative.No node formation was found after injecting nude mice with cell lysate or cellular DNA collected from equivalent amount of cells.It was indicated that the MDCK-G1 cells were of low-oncogenic potential.Conclusions The MDCK-G1 cell line could be used as a substrate to produce H5N1 influenza virus vaccine.

关 键 词：MDCK细胞 H5N1流感病毒 致瘤性和致癌性 

分 类 号：R392[医药卫生—免疫学]