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)

作　　者：张秋菊 杨雪 冯子祎 赵博 林洪娟 田丽媛 杨光 刘畅 李子莉 孙宏亮 ZHANG Qiuju;YANG Xue;FENG Ziyi;ZHAO Bo;LIN Hongjuan;TIAN Liyuan;YANG Guang;LIU Chang;LI Zili;SUN Hongliang(Changchun Institute of Biological Products Co.,Ltd.,Changchun 130012,Jilin Province,China)

机构地区：[1]长春生物制品研究所有限责任公司,吉林长春130012

出　　处：《中国生物制品学杂志》2024年第7期801-805,共5页Chinese Journal of Biologicals

基　　金：吉林省科技发展计划项目(20210401063YY)。

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 chromatograghy-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,IAM)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。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(without 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 SECHPLC,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 nano LC-MS/MS.Results Four proteins were successfully identified in the right peak 2 of SEC-HPLC main peak in lyophilized human rabies vaccine(Vero cell)stock solution(without human blood albumin),and the number of matching peptides 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 sequence coverage of the identified proteins ranged from 37.40%to 64.31%.A total of 280 peptides participated in the statistics,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 4a GV,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 

分 类 号：Q816[生物学—生物工程]