百日咳毒素S1亚基的基因脱毒  

Genetic detoxification of pertussis toxin S1 subunit

作　　者：刘宏博 朱德武 蔡梦瑶 马林 王芬 胡源 陈雯 付烈 段凯 LIU Hongbo;ZHU Dewu;CAI Mengyao;MA Lin;WANG Fen;HU Yuan;CHEN Wen;FU Lie;DUAN Kai(Wuhan Institute of Biological Products Co.,Ltd.,National Engineering Technology Research Center of Combined Vaccines,Vaccine Technology Innovation Center of Hubei Province,State Key Laboratory of Novel Vaccines for Emerging Infectious Diseases,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,国家联合疫苗工程技术研究中心,湖北省疫苗技术创新中心,新突发传染病新型疫苗研发全国重点实验室,湖北武汉430207

出　　处：《中国生物制品学杂志》2025年第2期143-148,154,共7页Chinese Journal of Biologicals

基　　金：国家“重大新药创制”科技重大专项资助项目(2019ZX09302059)。

摘　　要：目的对百日咳毒素(pertussis toxin,PT)-S1亚基进行基因编辑,以完成百日咳杆菌CS株的基因脱毒。方法制备百日咳杆菌CS株电转感受态细胞,电转导入携带Kana抗性基因的线性DNA重组片段,以两侧同源臂的重组与PT-S1亚基交换。用Kana抗生素筛选重组菌株,通过基因测序确定重组序列,并从重组菌株培养上清中获取PT蛋白,对其进行ELISA、Western blot及体外毒性分析。结果抗性筛选得到的重组菌株经基因组及mRNA测序,获得S1-R9K/E129G双突变菌株FE3和S1-R9K单突变菌株FE16。FE3和FE16株生长速率均略有降低,但最大菌浓度均上升,其摇瓶培养上清中PT蛋白含量分别可达796.8和185.9 ng/mL,PT蛋白可被S1亚基单克隆抗体1B7识别;FE3和FE16株PT蛋白体外毒性相比于PT标准品分别降低至0.0016%和0.0081%。结论采用电转线性DNA片段及Kana抗生素筛选的方法可对百日咳杆菌CS株进行基因编辑,获得PT蛋白毒性显著降低的基因脱毒菌株,为百日咳疫苗的进一步研究奠定了基础。Objective To edit the gene of pertussis toxin(PT)-S1 subunit in order to complete the genetic detoxification of Bordetella pertussis CS strain.Methods Electroporation competent cells of Bordetella pertussis CS strain were prepared and electro-transfected with the linear recombinant DNA fragments carrying Kana resistance genes,which was then exchanged with the PT-S1 subunit through recombination of the homologous arms on both sides.The recombinant strains were screened with Kana antibiotics,and the recombinant sequence was determined by gene sequencing.The PT protein was obtained from the culture supernatant of the recombinant strains for ELISA,Western blot and in vitro toxicity analysis.Results S1-R9K/E129G double mutant strain FE3 and S1-R9K single mutant strain FE16 were obtained by genome and mRNA sequencing.The growth rate of FE3 and FE16 decreased slightly,but the maximum bacterial concentration increased.The content of PT protein in shake flask culture supernatant of strain FE3 and FE16 was 796.8 and 185.9 ng/mL,respectively,and the PT proteins could be recognized by S1 subunit monoclonal antibody 1B7.The in vitro toxicity of PT proteins from strain FE3 and FE16 decreased to 0.0016%and 0.0081%,respectively,compared with that of PT standard.Conclusion Gene editing can be performed on Bordetella pertussis CS strain using electroporation of linear DNA fragments and Kana antibiotic screening,and result in genetically detoxified pertussis strains with PT protein of significantly reduced toxicity,which lays a foundation for further research on pertussis vaccines.

关 键 词：百日咳杆菌CS株 基因脱毒 百日咳毒素 1B7 CHO细胞凝集试验 

分 类 号：R378.42[医药卫生—病原生物学]