布鲁菌gntR4基因无痕缺失株构建及其生物学特性分析  

Construction and biological characterization of a non-marked gntR4 deletion mutant of Brucella abortus

作　　者：石文弢 尹伊 田明星 连拯民 成大荣[1] 于圣青[2] SHI Wentao;YIN Yi;TIAN Mingxing;LIAN Zhengmin;CHENG Darong;YU Shengqing(College of Veterinary Medicine,Yangzhou University,Yangzhou 225009,China;Shanghai Veterinary Research Institute,Chinese Academy of Agricultural Sciences,Shanghai 20024l,China)

机构地区：[1]扬州大学兽医学院,江苏扬州225009 [2]中国农业科学院上海兽医研究所,上海200241

出　　处：《扬州大学学报（农业与生命科学版）》2023年第2期1-9,共9页Journal of Yangzhou University：Agricultural and Life Science Edition

基　　金：国家自然科学基金资助项目(32072828、31972723);江苏现代农业(肉羊)产业技术体系建设专项[JATS(2018)306]。

摘　　要：布鲁菌是兼性胞内寄生菌,其入侵宿主细胞后的胞内存活能力决定着布鲁菌的毒力,布鲁菌毒力因子功能的研究对阐明其致病机制具有重要意义。在前期研究中利用转座子随机突变技术发现转录调控子GntR4与布鲁菌毒力相关,故进一步通过定向缺失方法构建布鲁菌gntR4基因无痕缺失株,并通过耐受性试验、免疫印迹试验、胞内存活试验、小鼠致病性试验等对gntR4缺失株的基本生物学特性进行评估。结果表明:gntR4缺失显著减弱布鲁菌抵抗阳性杀菌肽的能力,说明GntR4可能调控部分基因帮助布鲁菌抵抗宿主非特异性免疫杀伤。但GntR4与T4SS的表达无关,与布鲁菌在胞内的存活能力及对小鼠的致病力无关。综上,这一研究构建的基因无痕缺失方法为布鲁菌基因功能研究奠定基础,针对GntR4调控子的研究完善了GntR调控子家族的功能研究,为阐明布鲁菌抵抗宿主免疫杀伤与建立慢性感染机制间的关系提供依据。Brucella is a facultative intracellular parasite.Its ability to adapt to the host environment,survive intracellularly,and establish chronic infection determines the virulence of Brucella.Therefore,the exploration of virulence factors is helpful to clarify the specific process of bacterial survival in the host.The previous study used the signature-tagged mutagenesis and found that the transcriptional regulator GntR4 was related to the virulence of Brucella abortus.In this study,a non-marked gntR4 deletion mutant was constructed by the targeted gene deletion methods,the basic biological characteristics of gntR4 deletion mutant were evaluated by the sensitivity test,western blot,intracellular survival,and virulence assessment.The results showed that the gntR4 deletion mutant significantly reduced the ability of Brucella to resist positive antimicrobial peptides,indicating that GntR4 may regulate some unknown genes to facilitate Brucella to resist host non-specific immune killing.However,GntR4 had no concern with the expression of T4SS,the ability of Brucella abortus to survive intracellularly,and the pathogenicity of mice.In short,the gene deletion plasmid constructed in this study lays the foundation for the study of Brucella gene function,and the study of GntR4 regulators completes the functional study of the GntR regulator family,which provides a basis for clarifying the relationship between Brucella's resistance to immune killing and the establishment of chronic infection.

关 键 词：布鲁菌 转录调控子 基因缺失 免疫杀伤 

分 类 号：S852.61[农业科学—基础兽医学]