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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 and compare the main protein components of two inactivated enterovirus 71 (EV71) in clinical trials in China with mass spectrometry, and provide experimental evidence for improving the quality control method of EV71 vaccine. Methods Protein electrophoresis analysis was performed on two EV71 vaccine stocks (A, B stock solution) produced by Vero cells by gradient SDS-PAGE. The protein bands were recovered by gel filtration, and the protein samples were extracted by trypsin digestion. Mass spectrometry (LC / MS) of each band protein polypeptide recovered by secondary mass spectrometry sequence determination. Enzyme-linked immunosorbent assay (ELISA) and bicinchoninic acid method (BCA) were used to detect the concentration of EV71 antigen and the total protein in the concentrated vaccine liquid, respectively. Results The stock solution was concentrated more than 10 times and then analyzed by gradient SDS-PAGE. Antigen A was 4 bands and antigen B was 6 bands. Mass spectrometry was used to detect each band protein and search library analysis found that each band contains EV71 polyprotein; antigen A band 1, 3 and antigen B bands 1,3,4,5,6 contains monkey protein . The results of PSMs (number of identified peptides) found that the total protein ratio of EV71 protein was 96.7% and 95.0% respectively, and the specific activity was 865.5 and 291.8 U / μg. Conclusions Mass spectrometry can be used as an auxiliary method for protein detection in vaccines. Consideration should be given to limit requirements on the basis of multiple batches of tests to ensure the consistency of vaccine quality.