鼠伤寒沙门菌5'-nucleotidase基因缺失株的构建及其生物学特性的研究  

Construction of Δ5'-nucleotidase mutant strain of Salmonella typhimurium SL1344 and study on biological characteristics of the mutant strain

作　　者：张梦珂[1] 廖成水[1] 毛福超 王晓利[2] 张春杰[1] 李银聚[1] 吴庭才[1] 程相朝[1,3] ZHANG Meng-ke;LIAO Cheng-shui;MAO Fu-chao;WANG Xiao-li;ZHANG Chun-jie;LI Yin-ju;WU Ting-cai;CHENG Xiang-chao(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;Medical College ,Henan University of Science and Technology,Luoyang 471023,China;Luoyang Vocatiorud and Technical College ,Luoyang 471099,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,河南洛阳471023 [2]河南科技大学医学院,河南洛阳471023 [3]洛阳职业技术学院,河南洛阳471099

出　　处：《中国兽医科学》2019年第5期589-597,共9页Chinese Veterinary Science

基　　金：国家自然科学基金面上项目(31572489);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)

摘　　要：为了探究5'-nucleotidase基因的功能及其在鼠伤寒沙门菌感染中的作用,本研究首先构建缺失1 112 bp的5'-nucleotidase基因的重组自杀性质粒pR E 112Δ5'-nucleotidase,利用重组自杀性质粒介导的等位基因交换技术,两步法筛选出SL1344Δ5'-nucleotidase缺失株,并对其生物学特性进行初步研究。PCR及测序结果表明,SL1344Δ5'-nucleotidase构建成功,且回复株SL1344CΔ5'-nucleotidase构建成功。进一步研究表明,缺失株SL1344Δ5'-nucleotidase保留了亲本菌株SL1344的血清型1,4,5,12∶i∶1,2,且能够稳定遗传缺失1 112 bp的5'-nucleotidase基因,生长速度没有发生明显改变。但是,缺失株SL1344Δ5'-nucleotidase口服感染6周龄BALB/c小鼠的LD50为4.30×10~7CF U,毒力降低至亲本株SL1344的1.96%。免疫保护率试验结果显示,对6周龄BALB/c小鼠免疫后第17天,鼠伤寒沙门菌野生株攻毒有50%的保护率。缺失株免疫小鼠后第21天,血清抗体IgG水平达到最高。回复菌株与亲本亲株之间生物学特性无显著性差异,基本恢复到野生型水平。上述结果表明,SL1344Δ5'-nucleotidase基因缺失株构建成功,遗传稳定,毒力明显降低,且具有较好的免疫原性,为研究鼠伤寒沙门菌5'-nucleotidase基因与其致病力之间的关系提供了依据,并为研究它在鼠伤寒沙门菌感染中的作用奠定了基础。To explore the function of the 5’-nucleotidase gene and its role in Salmonella typhimurium infection,the SL1344Δ5’-nucleotidase mutant without 1 112 bp of Salmonella typhimurium recombinant suicide plasmid,and complemented mutant was constructed.Then,the biological characteristics of the mutant were detected. The results of PCR and sequencing showed that the SL1344Δ 5’-nucleotidase and the complemented strain were successfully constructed. The identification results suggested that the serotype of the strain was 1,4,5,12 ∶i∶1,2,identical to the parent SL1344 strain.In addition,the biochemical characteristics and growth rate did not significantly change and the strain was stable with the recombinant Δ5’-nucleotidase gene in vitro.Compared with the parent SL1344 strain,the virulence of the SL1344Δ5’-nucleotidase was attenuated to BALB/c mice.The mouse lethal test was 4.30×10^7 CFU,showing that the virulence of the strain was 1.98% of SL1344. The immunoprotective efficacy results showed that the protection rate of the animals infected with Salmonella typhimurium was 50% on the immunization by mutant strain.The serum antibodies peaked 21 st d post-immunization. The complemented strain was no significant difference in the biological characteristics with the parent SL1344 strain,and it basically returned to the wild-type level.The SL1344Δ5’-nucleotidase mutant was constructed successfully,and genetic stability,significantly reduced virulence.The above-mentioned results showed that 5’-nucleotidase plays an important role in regulating the virulence of SL1344,which provide a method for studying the relationship between the 5’-nucleotidase gene of Salmonella typhimurium and its pathogenicity,and lay a foundation for studying its role in Salmonella typhimurium infection.

关 键 词：鼠伤寒沙门菌SL1344 5’-nucleotidase基因 缺失株 回复株 生物学特性 

分 类 号：S852.612[农业科学—基础兽医学]