肠炎沙门菌eutR基因缺失株的构建及其部分生物学特性的鉴定  

Construction of a Salmonella enterica eutR gene-deficient strain and characterization of some of its biological properties

作　　者：赵桂新 段文龙 王凤杰 张晓禹 刘万 张鹿 陈钰彬 史秋梅[1] 吴同垒[1] ZHAO Guixin;DUAN Wenlong;WANG Fengjie;ZHANG Xiaoyu;LIU Wan;ZHANG Lu;CHEN Yu-bin;SHI Qiumei;WU Tonglei(Key Laboratory of Preventive Veterinary Medicine of Hebei Province,Hebei University of Science and Technology,Qinhuangdao,Hebei 066004,China)

机构地区：[1]河北科技师范学院河北省预防兽医学重点实验室,河北秦皇岛066004

出　　处：《中国兽医学报》2024年第11期2418-2423,共6页Chinese Journal of Veterinary Science

基　　金：河北省高等学校科学技术研究项目青年拔尖人才资助项目(BJK2022005);河北省现代农业产业技术体系创新团队肉牛疫病防控岗位资助项目(HBCT2023190201)

摘　　要：利用同源重组的方法成功构建了肠炎沙门菌eutR基因缺失株,通过对其生化特性、运动性、抵御体外应激作用以及在RAW264.7细胞内存活能力等方面进行研究。结果显示,肠炎沙门菌eutR基因缺失株生化特性和运动能力较肠炎沙门菌野生型没有明显变化;肠炎沙门菌eutR基因缺失株抵御酸、碱和抗氧化能力显著降低,而抵御热的能力无显著变化;eutR基因缺失株在RAW264.7细胞内存活能力较野生型显著降低。为了进一步分析eutR基因对肠炎沙门菌毒力因子表达的影响,利用SYBR Green法检测缺失株与野生株invH、ssav、ssrA、xthA、orf245、sodC、lrp、mrr1和hflk等毒力基因的相对表达水平,研究发现肠炎沙门菌eutR基因缺失株的上述毒力因子的表达较野生株均显著下调。经动物试验对肠炎沙门菌eutR基因缺失株进行LD50测定,结果显示eutR基因缺失株的LD50高于野生型,表明eutR基因可影响沙门菌的毒力。本研究阐明了eutR基因对肠炎沙门菌在巨噬细胞内存活能力、部分生物学特性及毒力的影响,为肠炎沙门菌基因工程减毒疫苗开发提供新的基因敲除靶标。The eutR gene deletion mutant of Salmonella enteritidis was successfully constructed by homologous recombination.Through the study of its biochemical characteristics,motility,resistance to stress in vitro and survival ability in RAW 264.7 cells,it was found that the biochemical characteristics and motility of the eutR gene deletion mutant of Salmonella enteritidis had no significant change compared with the wild type of Salmonella enteritidis.The ability of eutR gene deletion strain of Salmonella enteritidis to resist acid,alkali and oxidation was significantly reduced,while the ability to resist heat was not significantly changed;the survival ability of eutR gene deletion strain in RAW 264.7 cells was significantly reduced compared with the wild type.In order to further analyze the effect of eutR gene on the expression of virulence factors of Salmonella enteritidis,the relative expression levels of invH,ssav,ssrA,xthA,orf245,sodC,lrp,mrr1 and hflk virulence genes of the deletion strain and the wild strain were detected by SYBR Green PCR.It was found that the expression of the virulence factors mentioned above in the eutR gene deletion strain of Salmonella enteritidis was significantly down-regulated compared with that in the wild-type strain.The LD_(50)of the eutR gene-deleted strain of Salmonella enteritidis was determined by animal experiments,and the results showed that the LD_(50)of the eutR gene-deleted strain was higher than that of the wild-type strain,indicating that the eutR gene could affect the virulence of Salmonella.This study clarified the effect of eutR gene on the survival ability,some biological characieristics and virulence of Salmonella enteritidis in macrophages,and provided a new gene knockout target for the development of attenuated Salmonella enteritidis genetic engineering vaccine.

关 键 词：肠炎沙门菌 eutR基因 生物学特性 毒力因子 

分 类 号：S852.61[农业科学—基础兽医学]