质谱法分析肠道病毒71型疫苗原液的主要蛋白成分  

Analysis of major protein components of two domestic enterovirus 71 vaccine bulks by mass spectrometer in China

作　　者：高帆[1] 毛群颖[1] 张军楠[1] 秦文彦[2] 姚昕[1] 吴星[1] 王一平[1] 邵杰[1] 王轩[1] 徐苗[1] 梁争论[1] 

机构地区：[1]中国食品药品检定研究院 卫生部生物技术产品检定方法及其标准化重点实验室,北京100050 [2]北京博奥生物有限公司,北京102206

出　　处：《中国病毒病杂志》2013年第6期410-413,共4页Chinese Journal of Viral Diseases

基　　金：国家高技术研究发展计划(863计划)(2012AA02A402)

摘　　要：目的采用质谱法对我国2家进入临床试验肠道病毒71型(EV71)灭活疫苗的主要蛋白成分进行分析和比较,为完善EV71疫苗的质控方法提供实验依据。方法应用梯度SDS-PAGE法对2家生产自Vero细胞的EV71疫苗原液(A、B原液)进行蛋白电泳分析,蛋白条带经切胶回收、脱色、胰酶酶切后提取蛋白多肽,采用液质联用仪(LC/MS)对回收的各条带蛋白多肽进行二级质谱序列测定。采用酶联免疫法(ELISA)和二喹啉甲酸法(BCA)分别检测浓缩后的疫苗原液中EV71抗原含量和总蛋白含量,计算比活值。结果将原液浓缩10余倍后经梯度SDS-PAGE电泳分析,抗原A为4条带,抗原B为6条带。采用质谱法检测各条带蛋白并搜库分析发现,各条带均含有EV71多聚蛋白;抗原A条带1、3和抗原B的条带1、3、4、5、6含有猴源蛋白。经PSMs(鉴定多肽次数)计算发现EV71蛋白占总蛋白比例分别为96.7%和95.0%,比活值为865.5、291.8U/μg。结论质谱法可作为疫苗中蛋白检测的辅助方法,并考虑在多批次检测的基础上提出限量要求,为疫苗质量的一致性提供保障。Objective To analyze the major protein components of two domestic enterovirus 71 （EV71） vac- cines （A and B） manufactured in China that have entered clinical trials and provide reference for quality con- trol. Methods The EV71 bulks （antigen A and B） from Vero cells in two manufacturers were detected by gradient SDS-PAGE method. Protein bands were cut, extracted, de-colored, digested with restriction en- zymes, and then the peptides were extracted. Liquid chromatograph-mass spectrometer （LC/MS） was used to detect the secondary spectrum of the peptide sequence. The antigen content and total protein content from the concentrated bulk were measured by ELISA and BCA method to calculate the specific activity. Results The bulk was first concentrated about ten folds and analyzed by gradient SDS-PAGE which showed 4 bands for an- tigen A and 6 bands for antigen B. All bands contain EV71 polyprotein; band 1, 3 in antigen A and band 1, 3, 4, 5, 6 in antigen B contain monkey proteins. The proportions of EV71 protein to total proteins were 96.7% in antigen A and 95.0% in antigen B; and the specific activity was 865.5 and 291.8 in agtigens A and t3, respectively. Conclusions Mass spectrometer analysis may be used to detect residual host proteins and a threshold value should be set after evaluation of multiple batches for quality control.

关 键 词：肠道病毒71型(EV71) 疫苗 蛋白 质谱(MS) 

分 类 号：R373.2[医药卫生—病原生物学]