鼠伤寒沙门菌sseK3基因缺失株的构建及其生物学特性研究  

Construction and characterization of sseK3 mutant strain of Salmonella typhimurium

作　　者：杜付玉 廖成水(指导)[1] 郁川[1] 张春杰(指导)[1] 程相朝[1,2] 杨亚东 DU Fu-Yu;LIAO Cheng-Shui;YU Chuan;ZHANG Chun-Jie;CHENG Xiang-Chao;YANG Ya-Dong(College of Animal Science and Technology,Luoyang Key Laboratory of Live Carrier Biomaterial and Animal Disease Prevention and Control,Henan University of Science and Technology,Luoyang 471023,China)

机构地区：[1]河南科技大学动物科技学院,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 [2]洛阳职业技术学院,洛阳471003

出　　处：《中国免疫学杂志》2019年第19期2375-2380,共6页Chinese Journal of Immunology

基　　金：河南省自然科学基金项目(182300410078);国家自然科学基金项目(31572489和31802159);河南省科技攻关项目(182102110061);河南科技大学博士启动基金项目(13480071)资助

摘　　要：目的:检测sseK3基因的缺失对鼠伤寒沙门菌生物学特性的影响,为探究该基因的功能及开发安全有效的鼠伤寒沙门菌活疫苗提供了新思路。方法:通过重组自杀性质粒介导,两步筛选法敲除sseK 3基因并成功构建缺失菌株的回复菌株,进而检测其生物学和免疫学特性。结果:PCR、双酶切及测序结果证实成功构建了sseK3缺失株及其回复菌株。生物学特性研究表明,sseK3缺失菌株和亲本株SL1344血清型均为1,4,5,12:i:1,2,生化特性和生长速度无明显差异,且sseK3缺失株能够稳定遗传;sseK3缺失株口服感染6周龄BALB/c小鼠的LD 50为2.96×10^8 CFU,与亲本菌株SL1344相比,sseK3缺失株的LD 50值升高约1 035倍,毒力显著下降;免疫保护效力实验显示:sseK3缺失株免疫后第17天保护率为66.7%;在免疫小鼠后的第14天血清IgG达到最高水平;回复菌株与亲本亲株生物学特性无显著性差异,基本回复到野生型水平。结论:成功构建了鼠伤寒沙门菌sseK3基因缺失株,可稳定遗传,毒力显著降低,具有良好的免疫原性,推测sseK3对鼠伤寒沙门菌的毒力具有重要的调节作用,为探究该基因的功能及将其开发为更加安全有效的沙门菌弱毒疫苗候选菌株奠定了基础。Objective:It is a new idea to evaluate the biological characteristics of sseK3 mutant for the study of safe and effective live Salmonella typhimurium vaccine.Methods:The sseK3 gene was knocked out by recombinant suicide plasmid and the two-step screening method was used to successfully construct the restorer strain of the deleted strain,and then its biological and immunological properties were detected.Results:The results of PCR,dual-enzyme digestion and sequencing showed that sseK 3 and complemented mutant strain were successfully constructed.The identification results suggested that the serotype of the mutant was 1,4,5,12:i:1,2,identical to the parent SL1344 strain.In addition,biochemical characteristics and the growth rate did not significantly change and the mutant was stable with the recombinant sseK 3 gene in vitro.The sseK 3 deletion strain was used to infect 6-week-old BALB/c mice.The LD 50 was 2.96×10^8 CFU.And the sseK3 deletion strain compared with parents SL1344,the LD 50 value increased by approximately 1 035 times,which could conclude that the virulence of the sseK3 deletion strain decreased significantly.The immunoprotective efficacy results showed that the protection rate infected with of Salmonella typhimurium was 66.7%on the 17th day post-immunization.The serum antibody peaked 14 d post-immunization.The complemented mutant was no significant difference in the biological characteristics with the parent SL1344 strain,and it basically returned to the wild-type level.Conclusion:The sseK3 gene deletion strain of Salmonella typhimurium is successfully constructed,which was stable in inheritance,significantly reduced in virulence,and have good immunogenicity.It is speculated that sseK3 plays an important regulatory role in the virulence of Salmonella typhimurium whch laid a good foundation for study of the function of sseK3 gene and further development of a safer and more effective candidate strain of attenuated Salmonella vaccine.

关 键 词：鼠伤寒沙门菌SL1344 sseK 3基因 缺失株 回复菌株 生物学特性 

分 类 号：S852.612[农业科学—基础兽医学]