无动物源成分伤寒沙门菌Ty2株种子批传代稳定性研究  

Study on the genetic stability of the animal-free Salmonella typhi Ty2 strain

作　　者：张丽芝 张新庄 妥晓梅 刘爱萍 郑明睿 白贵杰 周海飞 罗树权 谭小梅 ZHANG Li-zhi;ZHANG Xin-zhuang;TUO Xiao-mei;LIU Ai-ping;ZHENG Ming-rui;BAI Gui-jie;ZHOU Hai-fei;LUO Shu-quan;TAN Xiao-mei(The First Research Department,Lanzhou Institute of Biological Products Co.,Ltd.,Center for Gansu Provincial Vaccine Engineering Research,Lanzhou 730046,Gansu Province,China)

机构地区：[1]兰州生物制品研究所有限责任公司第一研究室甘肃省疫苗工程技术研究中心,甘肃兰州730046

出　　处：《微生物学免疫学进展》2022年第6期32-40,共9页Progress In Microbiology and Immunology

摘　　要：目的检定无动物源成分伤寒沙门菌Ty2株主代种子及工作种子,验证工作种子连续传代30代次的传代稳定性,为伤寒Vi多糖蛋白结合疫苗无动物源工艺的建立提供前期研究数据。方法无动物源成分伤寒主代种子、工作种子及脱脂牛奶工作种子分别连续传代至第30代次,在《中华人民共和国药典》2020版(三部)(简称《中国药典》)伤寒沙门菌检定要求的基础上,进一步应用16S rRNA基因序列分析、多位点序列分型(multilocus sequence type,MLST),并以伤寒主要抗原表位Vi抗原片段中毒力基因viaB区域为参考基因组,比对连续传代伤寒工作种子基因序列的遗传稳定性。结果无动物源成分伤寒主代种子、工作种子及脱脂牛奶工作种子分别连续传代至第30代次种子,表型鉴定结果均符合《中国药典》伤寒沙门菌检定要求;连续传代至第30代次,Vi抗原血清凝集效价、O抗原血清凝集效价均未发生明显变化;16S rRNA基因序列鉴定PCR产物大小在1474~1478 bp之间,与NCBI数据库比对鉴定均为肠沙门菌肠亚种伤寒血清型;MLST分型结果表明,疫苗用伤寒沙门菌Ty2株为ST1型,连续传代30代次MLST序列分型保持一致;以伤寒Vi抗原片段中毒力基因viaB区域为参考基因组(GenebankD14156.1),伤寒主代种子及有、无动物源成分工作种子连续传代30代次,它们的viaB区基因序列均一致;它们与Genebank中viaB参考基因序列比对,均有3个单核苷酸多态性(single nucleotide polymorphism,SNP)。结论无动物源成分伤寒沙门菌Ty2株,经与脱脂牛奶株连续传代30代次比较,各项菌种检定结果均符合《中国药典》要求,相关基因性状保持一致且稳定,无动物源成分伤寒沙门菌Ty2株遗传稳定性良好,可用于疫苗的研发和生产。Objective To identify the master seed and working seed of Salmonella typhi Ty2 strain without animal origin components,and to verify the genetic stability of working seed consecutive 30 generations.Provide preliminary research data for Typhoid Vi-conjugate vaccine which is established of animal-free.Methods The animal-free master seed,working seeds and the skimmed milk seed were successively passed down to the 30th generation,based on the requirements of the 2020 edition of the Chinese Pharmacopoeia for the verification of Salmonella typhi,And further apply 16S rRNA gene sequencing analysis,multilocus sequence type(MLST),and used virulence gene viaB region of the Vi antigenic epitope of typhoid as the reference genome to compare the working seed genes genetic stability of sequences which is consecutive generations.Results The animal-free master seed,working seeds and the skimmed milk seed were successively passed down to the 30th generation seed.All tests were in line with the Chinese Pharmacopoeia Salmonella typhi test;with passed down to the 30th generation,the Vi antigen serum agglutination titer and O antigen-serum agglutination titer did not change significantly;the PCR product size identified by 16S rRNA gene sequence was between 1474-1478 bp,identified as Salmonella enterica enterica subspecies typhoid serotype by compared with NCBI.The results of MLST typing showed that the Ty2 strain of Salmonella typhi for vaccine was ST1 type,and the MLST sequence typing was consistent for 30 consecutive generations.Taking the viaB region of the virulence gene of the typhoid Vi antigenic fragment as the reference genome,the master seed of typhoid and the working seed with or without animal components were successively passaged for 30 generations,the gene sequences of the viaB region among them were consistent;when they were referenced with genebank No.D14156.1 Gene sequence alignment showed 3 single nucleotide polymorphisms(SNP).Conclusion The animal-free Salmonella typhi Ty2 strain has been compared with the skimmed m

关 键 词：伤寒沙门菌 遗传稳定性 多位点序列分型 基因测序技术 

分 类 号：R392[医药卫生—免疫学]