水痘减毒活疫苗Oka株在MRC-5细胞上的适应性及传代稳定性研究  

Adaptability and passage stability of live attenuated varicella vaccine Oka strain in MRC-5 cell

作　　者：张林亚 徐然 王玉霞 毛辉 高飞霞 何成 王亮 朱为 ZHANG Linya;XU Ran;WANG Yuxia;MAO Hui;GAO Feixia;HE Cheng;WANG Liang;ZHU Wei(Research Laboratory,Shanghai Institute of Biological Products Co.,Ltd.,Shanghai 200051,China)

机构地区：[1]上海生物制品研究所有限责任公司研发部,上海200051

出　　处：《微生物学免疫学进展》2024年第6期14-19,共6页Progress In Microbiology and Immunology

摘　　要：目的研究Oka株水痘病毒(the Oka varicella vaccine virus,V-Oka)在人二倍体细胞株MRC-5上的适应性和传代稳定性。方法将V-Oka株以不同感染复数(multiplicity of infection,MOI)接种MRC-5细胞,确定适宜MOI,同时将V-Oka株在MRC-5细胞上连续传代培养至42代,测定病毒滴度和核酸拷贝数,并采用二代测序技术对32、34、36和38代毒株进行全基因组测序,分析各代次毒株疫苗型位点检出率(proportion of vaccine-type allele,Pv)≥30%的40个单核苷酸多态性(single nucleotide polymorphism,SNP)位点的变化趋势。结果V-Oka株在MRC-5细胞上培养的最适MOI为0.0040。V-Oka株在MRC-5细胞上连续传代10代内病毒增殖特性稳定,平均病毒滴度水平为(5.50±0.16)lgPFU/mL,平均核酸拷贝数水平为(10.99±0.15)lgcopies/mL。二代测序技术测序发现38代以内V-Oka株在上述40个SNP位点的遗传特征保持稳定。结论V-Oka株在MRC-5细胞上传代至38代,增殖稳定性和遗传稳定性良好。Objective To investigate the adaptability and passage stability of the Oka varicella vaccine virus(V-Oka) when passaged onto the human diploid cell line MRC-5. Methods The V-Oka strain was inoculated into MRC-5 cells with various multiplicity of infection(MOI) to determine the optimal MOI. Subsequently, the V-Oka strain was continuously subcultured in MRC-5 cells up to passage 42. Virus titers and nucleic acid copies were determined. Whole-genome sequencing of the strains at passages 32,34,36,and 38 were performed using next-generation sequencing(NGS) technology, as well as analyzing the variation trend of 40 single nucleotide polymorphism(SNP) loci with a proportion of vaccine-type allele(Pv) ≥30% in each passage. Results The optimal MOI for culturing the V-Oka strain in MRC-5 cell was determined to be 0.004 0. Following 10 consecutive passages in MRC-5 cell, the proliferation of the V-Oka strain was stable, with an mean virus titer of(5.50±0.16) lgPFU/mL,and an average nucleic acid copy number of(10.99±0.15) lgcopies/mL. Additionally, NGS analysis revealed that the genetic characteristics of the 40 SNPs remained stable within 38 passages of the V-Oka strain. Conclusion These results indicate that the proliferation and genetic characteristics of the V-Oka strain remained stable after being subcultured in MRC-5 cell up to passage 38.

关 键 词：水痘减毒活疫苗Oka株 MRC-5细胞 适应性 传代稳定性 二代测序 

分 类 号：Q93-3[生物学—微生物学]