百日咳疫苗用菌株基因的遗传稳定性研究  

Genetic stability of pertussis vaccine strains

作　　者：顾琴[1] 梁疆莉[1] 高娜[1] 马艳[1] 姬秋彦[1] 史荔[1] 孙明波[1] GU Qin;LIANG Jiang-li;GAO Na;MA Yan;JI Qiu-yan;SHI Li;SUN Ming-bo(The Joint Laboratory of Institute of Medical Biology,Chinese Academy of Medical Science&Peking Union Medical College,Kunming 650118,Yunnan Province,China)

机构地区：[1]中国医学科学院北京协和医学院医学生物学研究所联合实验室

出　　处：《微生物学免疫学进展》2019年第6期1-7,共7页Progress In Microbiology and Immunology

基　　金：中国医学科学院医学与健康科技创新工程(2016-I2M-1-019);重大新药创制科技重大专项基金资助项目(2015ZX09101031);云南省科技厅科技计划项目(2016GA004-2019HC006)

摘　　要：目的对百日咳疫苗(pertussis vaccine)用菌株的主要保护性抗原基因序列的遗传稳定性进行研究,进一步验证此前一代测序的结果,为疫苗的安全性和有效性提供依据。方法采用第二代测序技术(next generation sequencing,NGS)对百日咳疫苗生产用菌株的主代代次(P4)、工作代次(P7)、疫苗代次(P13)及疫苗代次后连续传3代的第3代样品V3代次(P16)的主要抗原基因序列进行测序并分析。采用ELISA和Lowry法对百日咳疫苗生产用菌株各代次主要抗原组分进行抗原含量检测及比活性检测。结果百日咳毒素(pertussis toxin,PT)S1亚基上的8个等位基因氨基酸相对应的24个碱基位点中最高突变率为0.38%,其中疫苗代次最高突变位点(A 2789 C 0.92%)在S5亚基上,其他各代次最高突变位点均不在S亚基上。PT全基因组最高突变率<4%,且同一个位点不同代次的碱基突变呈不规律性,突变率没有随传代次数的增加而增高。百日咳黏附素(pertactin,PRN)的24个抗原表位中最高突变率在疫苗代次T 951 G位点,突变率为1.66%;其他位点的突变率均<0.8%。全基因组中突变率最高的位点为V3代次C 2025 T,突变率为6.24%;各代次PRN全基因组最高突变率<6.24%,且不在主要抗原表位区域内;在同一个位点不同代次的碱基突变没有随传代次数的增加而增高,呈不规律性。丝状血凝素(filamentous hemagglutinin,FHA)中B细胞抗原表位区中碱基最高突变率为0.98%(V3代次T 6449 G位点),其他代次均<0.8%;全基因组各代次位点最高突变率为V3代次C 7323 G,突变率为9.89%;其余代次突变率均<9.89%,且不在抗原表位区,同一个位点不同代次的碱基突变没有随传代次数的增加而增高,呈不规律性。PT的ELISA比活性平均值为0.532±0.100,最低限值为0.232。PRN的ELISA比活性平均值为0.885±0.110,最低限值为0.555。FHA的ELISA比活性平均值为0.701±0.170,最低限值为0.191,百日咳菌种抗原含量和比活性Objective To study the genetic stability of major protective antigen gene sequences and to verify the sequencing results of parent strain,which will provide a basis for the safety and effectiveness of the vaccine.Methods The main antigen gene sequences of pertussis vaccine strains from the master cell bank(P4),working cell bank(P7),products(P13)and the third-generation samples V3(P16)from the production strain,were sequenced and analyzed by using next generation sequencing,NGS.The ELISA and Lowry methods were used to detect the antigen content and specific activity of the main antigen components of these pertussis vaccine strains.Results The highest mutation rate of the 24 bases corresponding to the 8 amino acids on the S1 subunit of pertussis toxin(PT)PT was 0.38%,and the highest mutation site of vaccine strains(A 2789 C 0.92%)was on S5 subunit,and the highest mutation site of other generation strains was not on S subunit.The highest mutation rate of the whole genome of PT was less than 4%,and the base mutations of different generations at the same site were in irregular,and the mutation rate did not increase with the number of passages.The highest mutation rate among the 24 epitopes of persactin(PRN)was at the T951G site of the vaccine,and the mutation rate was 1.66%;the mutation rate of other sites was<0.8%.The highest mutation rate in the whole genome was at C2025T of V3 strain,and the mutation rate was 6.24%;the highest mutation rate of PRN gene of each generation was<6.24%,and it was not in the main epitope region;and the base mutation of different generations in the same locus did not increase with the number of passages,which was not in regular.The highest base mutation rate in the B cell epitope region in filamentous hemagglutinin(FHA)was 0.98%(V3 T6449G locus),and the mutation rate of other sites were less than 0.8%;the highest mutation rate in the whole genome was at C7323G of V3 strain,and the mutation rate was 9.89%,while the mutation rates of other generation are less than 9.89%,and the base mutatio

关 键 词：二代测序 百日咳 抗原表位 突变率 遗传稳定性 

分 类 号：R378.4[医药卫生—病原生物学]