麻疹病毒长-47纯化株(2BS细胞)毒种库的建立  

Establishment of purified seed bank of measles virus Chang-47 strain(2BS cells)

作　　者：沈红杰 张宇 黄果勇[2] 李海燕 闫磊 王玮 郑晓丽 

机构地区：[1]长春祈健生物制品有限公司,吉林长春130012 [2]广西壮族自治区疾病预防控制中心,广西南宁530000

出　　处：《中国生物制品学杂志》2015年第12期1339-1343,共5页Chinese Journal of Biologicals

摘　　要：目的建立以人胚肺二倍体细胞（2BS）为基质的麻疹病毒长-47纯化株毒种库。方法将麻疹病毒长-47纯化株毒种（CEC）主代主种子批适应于2BS细胞,传至第4代和第8代,分别作为主种子批及工作种子批,对毒种进行鉴别试验、无菌试验、支原体检查、外源因子检查和病毒滴度检测,并对主种子批毒种进行猴体神经毒力试验和猴体免疫原性检测。将毒种连续传代,观察各代次病毒的收获时间,并测定病毒滴度,分析毒种的传代稳定性;将主种子批和工作种子批分别置于-20和-70℃保存,不同时间取样,检测病毒滴度,分析毒种保存温度稳定性;选取4个代次毒种（D4、D8、D15、D19）提取病毒核酸,扩增H基因序列,分析H基因在传代过程中的遗传稳定性。结果主种子批和工作种子批毒种鉴别试验、无菌试验、支原体及外源因子检查均符合《中国药典》三部（2010版）要求,病毒滴度分别为5.88和6.00 log CCID50/ml,主种子批毒种具有良好的安全性及免疫原性。麻疹病毒长-47纯化株毒种适应于2BS细胞后,收获病毒时间一般在6-7 d,病毒滴度稳定在5.5-6.5 log CCID50/ml之间。主种子批和工作种子批病毒于-20和-70℃保存10个月,病毒滴度无明显变化。D4-D19代病毒无核苷酸发生改变,与麻疹病毒Chang-47（Gen Bank登录号：EF033071）比对结果显示,D4-D19代病毒核苷酸序列21 nt由G→C,属于同义突变,同源性达99.9%。结论建立了麻疹病毒长-47纯化株（2BS）毒种库,并按照《中国药典》三部（2010版）要求进行了全面检定,为制备以人胚肺二倍体细胞为基质的麻疹减毒活疫苗奠定了基础。Objective To establish a seed bank of measles virus Chang-47 strain using human diploid cells（2BS） as a matrix. Methods Purified virus seeds（CEC） from master seed lot of measles virus Chang-47 strain was subcultured in2 BS cells to passages 4 and 8, and used as master and working seeds respectively. The seeds were tested for identity,sterility, mycoplasma, adventitious agents, and titer, while the master seeds for neurovirulence and immunogenicity in monkeys. The virus seeds were further subcultured, of which each passage was observed for harvest time, determined for titer and analyzed for stability. The master and working seeds were stored at-20 and-70 ℃ separately, from which samples were taken at various time points and determined for titer to evaluate the stability of virus seeds after storage.Nucleic acids were extracted for virus seeds of four passages（D4, D8, D15 and D19）, from which H gene was amplified and analyzed for genetic stability during subculture. Results The test results of identity, sterility, mycoplasma and adventitious agents of master and working seeds met the requirements in Chinese Pharmacopoeia（Volume Ⅲ, 2010edition）, of which the virus titers were 5. 88 and 6. 00 log CCID50/ ml respectively. Both the seeds showed good safety and immunogenicity. After adaption in 2BS cells, the harvest time of Chang-47 strain was 6 - 7 d in general, of which the titer was as stable as 5. 5 - 6. 5 log CCID50/ ml. Neither the virus titer of master and working seeds showed significant change after storage at-20 and-70 ℃ for 10 months. No changes of nucleic acids were observed in virus seeds of four passages.However, the nucleotide at site 21 changed from G to C as compared with that of Chang-47 strain in Gen Bank（EF033071）, which belonged to sense mutation, indicating a homology of 99. 9%. Conclusion The seed bank of purified measles virus Chang-47 strain（2BS cells） was established and subjected to overall control tests according to the requirements in Chinese Pharmacopoeia（Vo

关 键 词：麻疹病毒长-47纯化株 人二倍体细胞 毒种库 基因稳定性 

分 类 号：R373.11[医药卫生—病原生物学] R392-33[医药卫生—基础医学]