论文部分内容阅读
目的:研究四价流感病毒裂解疫苗生产用毒种的传代稳定性，确保毒种适用于疫苗生产。方法:将WHO推荐的季节性流感疫苗甲型H1N1和H3N2、乙型BV和BY病毒株(原始种子批)在鸡胚中传代扩增，制备生产用主种子批和工作种子批毒种。按中国药典2015年版三部的要求对毒种进行检定，包括鉴别试验、病毒滴度和血凝滴度测定、外源微生物检查等。将毒种连续传10代，用反转录PCR扩增第2、3、4、5、10代病毒鸡胚尿囊液中的血凝素和神经氨酸酶(neuraminidase，NA)的基因片段，并进行序列测定，分析传代过程中血凝素和NA的核苷酸和氨基酸序列同源性。结果:主种子批和工作种子批毒种的抗原性均与推荐的病毒株一致。各型病毒滴度均>6.5 lg半数鸡胚感染量/ml，血凝滴度均≥1∶160。2个种子批的支原体和无菌检查结果均为阴性，主种子批外源性禽白血病病毒和禽腺病毒检查结果亦为阴性。毒种在鸡胚中连续传代后，各型病毒株血凝素和NA的核苷酸序列同源性均>99%，氨基酸序列同源性均为100%。结论:四价流感病毒裂解疫苗生产用毒种具有良好的传代稳定性，可用于疫苗生产。“,”Objective:To study the passage stability of virus seeds used for quadrivalent influenza virus-split vaccine to ensure their application in vaccine production.Methods:The influenza A (H1N1 and H3N2) and influenza B (BV and BY) virus strains (original seed lot) recommended by WHO for seasonal influenza vaccine were passed and amplified in chicken embryos to prepare the virus seeds of master seed lot and working seed lot. Then the virus seeds were verified according to Chinese pharmacopoeia 2015 (Volume Ⅲ), including identification test, virus titer and hemagglutination titer detections, and exogenous microorganism examination. Virus seeds were continuously passed to the 10th generation. The hemagglutinin (HA) and neuraminidase (NA) gene fragments of the 2nd, 3rd, 4th, 5th, and 10th generation viruses from chicken embryo allantoic fluid were amplified with reverse transcription-PCR and sequenced to analyze the nucleotide and amino acid sequence homology during passage.Results:The antigenicity of master seed lot and working seed lot viruses was consistent with the virus strains recommended. All type virus titers were >6.5 lg 50% egg infective dose/ml, and the hemagglutination titers were ≥1∶160. Mycoplasma and sterility test results were negative in both seed lots. Exogenous avian leukosis virus and avian adenovirus were also negative in master seed lot. The HA and NA nucleotide sequence homology was >99% and amino acid sequence homology was 100% after the virus strains were consecutively passed in chicken embryos.Conclusion:The virus seeds of quadrivalent influenza virus-split vaccine have good passage stability and can be used for vaccine production.