百日咳鲍特菌菌毛蛋白纯化方法的建立及免疫原性的初步评价  

Purification and immunological evaluation of Bordetella pertussis fimbriae

作　　者：杨柏峰 田聪 朱德武 潘聪 胡太平 施斌 王丽 周昉 杨晓明 YANG Bai-feng;TIAN Cong;ZHU De-wu;Pan Cong;HU Tai-ping;SHI Bin;Wang Li;ZHOU Fang;YANG Xiao-ming(Bacterial Vaccine Department of Research,Wuhan Institute of Biological Products Co.,Ltd.,National Engineering Technology Research Center of Combination Vaccine,Wuhan 430207,Hubei Province,China)

机构地区：[1]武汉生物制品研究所有限责任公司细菌类疫苗研究室,国家联合疫苗工程技术研究中心,湖北武汉430207 [2]中国生物技术股份有限公司,北京100029

出　　处：《微生物学免疫学进展》2021年第2期35-42,共8页Progress In Microbiology and Immunology

基　　金：国家“重大新药创制”科技重大专项资助项目(2018ZX09738003)。

摘　　要：目的建立一种适合生产放大的百日咳鲍特菌菌毛蛋白(fimbriae, Fim)纯化新工艺,并对其免疫原性进行初步评价。方法百日咳黏附素(pertactin, PRN)流穿液经一步盐析和一步复合型阴离子Capto Adhere疏水层析流穿法进行纯化,以纯度和回收率为检测指标,对硫酸铵盐析质量体积比和疏水离子层析条件进行优化,进行连续3批生产验证工艺稳定性。检验精纯Fim蛋白毒性,将合格Fim蛋白与百日咳其他组分配伍成五组分无细胞百日咳疫苗(acellular component pertussis vaccine, acPV),免疫小鼠,评价免疫原性。结果成功建立了从PRN流穿液中纯化Fim蛋白的方法。该工艺制备的3批Fim蛋白纯度(>95%)高,回收率均在60%以上,具有良好的重复性,且Fim蛋白符合特异性毒性试验要求。5μg Fim蛋白免疫小鼠后在7、28、35和42 d血清中的抗Fim抗体几何平均滴度(geometric mean titer, GMT)分别为10.6、97.7、102.9、192.6 IU/mL,与生理盐水组相比具有统计学差异(P<0.05)。结论利用QbD理念建立了适合生产放大的百日咳鲍特菌菌毛蛋白纯化新工艺,且蛋白具有良好的免疫原性,未来有望作为组分百日咳疫苗的候选抗原。Objective To develop a novel purification method that is used for scale-up of pertussis fimbriae(Fim) protein purification, and to evaluate its immunogenicity. Methods Pertussis pertactin(PRN) flow-through liquid was purified through one-step salting-out and Capto Adhere hydrophobic ion chromatography. Moreover, the concentrations(mass to volume ratio) of ammonia sulfate in step 1 and the conditions of hydrophobic anion exchange chromatography were optimized, and the purity and recovery rate were used as the process control index. To verify the stability of the process, three process validation batches were conducted. The toxicity test for pure Fim protein was also carried out, and the five-component acellular component pertussis vaccine(acpv) were prepared by combining the qualified Fim protein with other components of pertussis. The immunogenicity was evaluated after immunizing mice. Results A novel purification process for Fim protein from PRN flow-through was developed. The purity of three batches of Fim protein obtained by this process was high(>95%), the recovery rate of which was over 60% and had good repeatability. Fim protein passed the specific toxicity test as well. The geometric mean titer(GMT) of anti-Fim antibody in the serum which was collected at days 7, 28, 35 and 42 after immunizing with 5 μg Fim protein were 10.6 IU/mL, 97.7 IU/mL, 102.9 IU/mL and 192.6 IU/mL, respectively, which was statistically significant difference(P<0.05) compared with normal saline group. Conclusion A new purification method which is suite for scale-up of pertussis fimbriae(Fim) protein purification was developed by using the concept of QbD. The Fim protein has good immunogenicity, which is expected to be a candidate antigen of pertussis vaccine in the future.

关 键 词：百日咳 百日咳黏附素 菌毛蛋白 盐析 疏水层析 抗Fim抗体 

分 类 号：R516.6[医药卫生—内科学]