冻干人用狂犬病疫苗(Vero细胞)原液(无人白)体积分子排阻-高效液相色谱主峰右侧峰2蛋白鉴定
Identification of peak 2 protein on right side of main peak of SEC-HPLC in lyophilized human rabies vaccine(Vero cells)stock solution(without human blood albumin)
张秋菊 1杨雪 1冯子祎 1赵博 1林洪娟 1田丽媛 1杨光 1刘畅 1李子莉 1孙宏亮1
作者信息
- 1. 长春生物制品研究所有限责任公司,吉林长春 130012
- 折叠
摘要
目的 对冻干人用狂犬病疫苗(Vero细胞)原液(无人白)体积分子排阻-高效液相色谱(size exclusion chro-matograghy-high performance liquid chromatography,SEC-HPLC)主峰右侧峰2蛋白进行鉴定,以明确该峰的蛋白成分,保证疫苗质量.方法 将冻干人用狂犬病疫苗(Vero细胞)原液(无人白)进行SEC-HPLC分析,收集主峰右侧峰2,经超滤浓缩后,进行二硫苏糖醇(dithiothreitol,DTT)还原、碘代乙酰胺(iodoacetamide,I AM)烷基化和胰蛋白酶(Trypsin)酶解,产物进行nanoLC-MS/MS串联质谱分析.结果 冻干人用狂犬病疫苗(Vero细胞)原液(无人白)SEC-HPLC 主峰右侧峰2成功鉴定4个蛋白,单个蛋白匹配肽段数在59～79个之间;单个蛋白匹配特征肽段种数在6～12种之间;单个蛋白匹配特征肽段检出次数在20～36次之间;鉴定蛋白的序列覆盖率在37.40％～64.31％之间.参与统计的肽段共280个,质谱偏差均小于0.15 Da.结论 冻干人用狂犬病疫苗(Vero细胞)原液(无人白)SEC-HPLC主峰右侧峰2均来源于狂犬病病毒株4aGV,为疫苗颗粒解离物.
Abstract
Objective To identify the peak 2 protein on the right side of the main peak of size exclusion chromatograghy-high performance liquid chromatography(SEC-HPLC)in freeze-dried human rabies vaccine(Vero cell)stock solution(with-out human blood albumin),so as to determine the protein composition of the peak and ensure the vaccine quality.Methods The lyophilized human rabies vaccine(Vero cells)stock solution(without human blood albumin)was analyzed by SEC-HPLC,of which the peak 2 on the right side of the main peak was collected and concentrated by ultrafiltration,then reduced by dithiothreitol(DTT),alkylated by iodoacetamide(IAM)and hydrolyzed by Trypsin.The products were analyzed by nanoLC-MS/MS.Results Four proteins were successfully identified in the right peak 2 of SEC-HPLC main peak in lyophi-lized human rabies vaccine(Vero cell)stock solution(without human blood albumin),and the number of matching pep-tides for a single protein ranged from 59 to 79.The number of single protein-matching characteristic peptides ranged from 6 to 12.The detection times of single protein-matching characteristic peptide segments ranged from 20 to 36 times.The sequ-ence coverage of the identified proteins ranged from 37.40％ to 64.31％.A total of 280 peptides participated in the statis-tics,and the mass spectrum deviation was less than 0.15 Da.Conclusion The peak 2 on the right side of SEC-HPLC main peak of lyophilized human rabies vaccine(Vero cell)stock solution(without human blood albumin)is derived from rabies strain 4aGV,which is the vaccine particle dissociation.
关键词
狂犬病疫苗(Vero细胞)原液(无人白)/狂犬病病毒株4aGV/体积分子排阻-高效液相色谱/蛋白鉴定/nanoLC-MS/MS分析/疫苗病毒颗粒Key words
Rabies vaccine(Vero cells)stock solution(without human blood albumin)/Rabies strain 4aGV/Size exclu-sion chromatograghy-high performance liquid chromatography(SEC-HPLC)/Protein identification/nanoLC-MS/MS anal-ysis/Vaccine virus particle引用本文复制引用
基金项目
吉林省科技发展计划项目(20210401063YY)
出版年
2024
NETL
NSTL
万方数据