减毒鼠伤寒沙门菌SL1344ΔsseK1Δasd宿主-载体平衡致死系统的构建及其生物学特性研究  

Construction and characterization of host-vector balanced lethal system of attenuated Salmonella typhimurium SL1344ΔsseK1Δasd

作　　者：杨亚东[1] 丁轲[1] 张春杰[1] 程相朝[1] 贾艳艳[1] 何雷[1] 

机构地区：[1]河南科技大学动物疫病与公共卫生重点实验室/洛阳市活载体生物材料与动物疫病防控重点实验室,洛阳471023

出　　处：《中国免疫学杂志》2017年第10期1521-1526,共6页Chinese Journal of Immunology

基　　金：河南省科技攻关项目(112102110019)资助

摘　　要：目的:为构建减毒鼠伤寒沙门菌分泌性蛋白K1缺失株平衡致死系统,并将其开发为能够稳定携带外源基因的活疫苗载体。方法:利用重组自杀性质粒(pREΔasd)介导的等位基因交换技术,以减毒鼠伤寒沙门菌株SL1344Δsse K1为亲本株,运用二步法筛选asd基因缺失株SL1344Δsse K1Δasd。将携带有asd基因的无抗性p YA3493质粒电转至上述缺失菌株SL1344Δsse K1Δasd,构建SL1344Δsse K1Δasd(p YA3493)重组菌株。结果:PCR及测序结果表明SL1344Δsse K1Δasd(p YA3493)构建成功。进一步研究表明重组菌株SL1344Δsse K1Δasd(p YA3493)血清型和强毒株SL1344及亲本株SL1344Δsse K1相同,且能稳定遗传缺失后的asd基因;其生化特性和生长速度与强毒株SL1344及亲本株SL1344Δsse K1相比均无明显差异。小鼠毒力试验表明,SL1344Δsse K1Δasd(p YA3493)口服感染6周龄BALB/c小鼠的LD50为5.24×108CFU,毒力较强毒株SL1344约下降至0.048%;免疫保护效力试验显示,运用鼠伤寒沙门菌野生毒株对免疫后第17天的小鼠攻毒有62.5%的保护率,与亲本株SL1344Δsse K1基本一致。结论:以上结果表明,鼠伤寒沙门菌分泌性蛋白K1缺失株宿主-载体平衡致死系统构建成功,且遗传稳定,毒力明显降低,具有较好的免疫原性,为深入研究以鼠伤寒沙门菌为载体的口服多价疫苗奠定基础。Objective： To develop a host-vector balanced lethal system of attenuated Salmonella typhinurium secreted effector K1 mutant,and an live vaccine vector which stably carries exogenous genes. Methods： We constructed SL1344Δsse K1Δasd mutant strain by recombinant suicide plasmid（ pREΔasd）,and screened by two-step method,transformed p YA3493 plasmid containing the asd gene without resistance electric into the mutant strain of SL1344Δsse K1Δasd,then the recombinant strain SL1344Δsse K1Δasd（ p YA3493） was constructed successfully. Results： The results of PCR and sequencing showed that SL1344Δsse K1Δasd（ p YA3493） was constructed successfully. Further studies had shown that the serotype of the recombinant strain was identical to the parent SL1344Δsse K1 and wild SL1344 strains,and the mutant was stable with the recombinant Δasd gene in vitro. It was found that the recombinant strain had displayed identical growth profile and biochemical characteristics compared with the parent SL1344Δsse K1 strain and wild SL1344 strain. The oral challenge of bacteria in mice revealed that LD50 of the recombinant strain was 5. 24×108CFU,and the toxicity reduced to about 0. 048%; the immunoprotective effect assay showed that the protection rate infected with wild strain of Salmonella typhimurium was 62. 5% on the 17 th day post-immunization,which was identical to the parent SL1344Δsse K1. Conclusion：These results show that the secreted effector K1 gene deleted mutant host-vector balanced lethal system of Salmonella typhimurium SL1344 strain has been successfully constructed,and genetic stability,significantly reduced virulence,which has laid a foundation for developing potential oral live vaccin vector to express foreign genes.

关 键 词：鼠伤寒沙门菌 自杀性质粒 宿主-载体平衡致死系统 活疫苗载体 

分 类 号：S852.612[农业科学—基础兽医学]