鼠伤寒沙门菌SL1344株分泌性蛋白K1缺失株的构建及其生物学特性的初步研究  

Construction and characterization of secreted effector K1 gene deleted mutant of Salmonella typhimurium SL1344 strain

作　　者：杨亚东[1,2] 张春杰 程相朝[1,2] 丁轲 李银聚[1,2] 郁川 廖成水[1,2] 陈松彪 王二鑫[1,2] 张晓洁 

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,河南洛阳471023 [2]洛阳市活载体生物材料与动物疫病防控重点实验室,河南洛阳471023

出　　处：《中国兽医科学》2017年第9期1087-1093,共7页Chinese Veterinary Science

基　　金：河南省科技攻关项目(112102110019)

摘　　要：为研制更加安全有效且遗传背景清晰的鼠伤寒沙门菌(S.typhimurium)疫苗弱毒株,本研究首先构建了含缺失1 011 bp sseK1基因的重组自杀性质粒PREΔsseK1,利用重组自杀性质粒介导的等位基因交换技术,两步法筛选SL1344的sseK1缺失株。PCR、双酶切及测序结果表明SL1344ΔsseK1构建成功。生物学特性初步研究表明,与亲本菌株SL1344相比,缺失株SL1344ΔsseK1保留了亲本株SL1344的血清型1,4,5,12∶i∶1,2,且能够稳定遗传缺失后的sseK1基因,生长速度及生化特性没有发生明显改变。小鼠毒力试验表明,SL1344ΔsseK1缺失株口服感染6周龄BALB/c小鼠的LD_(50)为6.63×10~8CFU,毒力较亲本株SL1344下降至0.048%。免疫保护效力试验显示,小鼠免疫后第14天血清抗体Ig G含量达到最高,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。以上研究结果表明,SL1344株sseK1基因缺失株构建成功,且遗传稳定,毒力明显降低,具有较好的免疫原性,为研究sseK1基因的致病机制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。To develop safer and more effective alive attenuated Salmonella typhimurium vaccines,the recombinant suicide plasmid （PREΔsseK1） without 1 011 bp sseK1 gene was built by homologous recombination technology in this study.The sseK1 gene deleted mutant of Salmonella typhimurium SL1344 strain was screened using two-step method by recombinant suicide plasmid-mediated allelic exchange technology.The results of PCR,dual-enzyme digestion and sequencing showed that the SL1344ΔsseK1 strain was successfully constructed.It was found that the serotype of the mutant was 1,4,5,12 ∶ i ∶ 1,2,identical to the parent SL1344,and the mutant was stable with the recombinant ΔsseK1 gene in vitro.In addention,we found that the sseK1 deletion mutant displayed identical growth profile and biochemical characteristics compared with the parent SL1344 strain.The mutant strain showed obvious changes in its virulence and oral challenge of bacteria in mice revealed that LD（50） of the mutant strain was 6.63×10-8 CFU,the toxicity being reduced about 0.048%.The immunoprotective effect assay showed that the serum IgG antibody could achieve the highest leverl on day 14 post-immunization,and the protection rate infected with wild strain of Salmonella typhimurium was 62.5% on day 17 post-immunization.In conclusion,the SL1344ΔsseK1 mutant was constructed successfully,and genetic stability,and significantly reduced virulence,which laid a foundation for studies of the pathogenesis of sseK1 gene and further development of a safer and more effective candidate strain of attenuated Salmonella vaccines.

关 键 词：鼠伤寒沙门菌SL1344 重组自杀性质粒 sseK1基因 生物学特性 

分 类 号：S855.99[农业科学—临床兽医学]