摘要
旨在利用结核分枝杆菌牛变种C68001菌株的突变体文库,高通量筛选鉴定影响生物膜形成的关键基因。本研究利用结核分枝杆菌牛变种C68001菌株的突变体文库,用苏通培养基进行培养,筛选生物膜表型差异明显的突变株,通过抗性标记挽救法及测序,确定突变基因位置;检测突变基因对菌株抗氧化性及抗SDS性能的影响;进一步以噬菌体系统构建靶标基因缺失、回补及过表达菌株,利用扫描电子显微镜检测靶基因对细菌菌丝影响,通过测定细菌的抗酸、抗氧化、抗SDS以及在小鼠巨噬细胞系J774A.1中的存活性能,检测靶标基因对细菌抗逆性和胞内存活的影响。利用突变体文库,筛选到8个生物膜形成缺陷的菌株,鉴定其中4个为单基因突变菌株,突变基因分别为Rv1096、Rv3425、Rv 3136和Rv 3671c;构建Rv 3671c基因敲除、回补和过表达菌株,通过PCR及Western blot验证构建成功,测试其生物膜形成能力、抗逆性和胞内存活性能,发现Rv 3671c基因可提高结核分枝杆菌牛变种抗SDS、生物膜形成及胞内存活等方面的能力。Rv 3671c基因在结核分枝杆菌牛变种C68001的生物膜形成和抗逆性能力中发挥重要作用。构建的结核分枝杆菌牛变种C68001菌株突变体文库可高效用于靶标基因的筛选和鉴定,为探索结核分枝杆菌牛变种的基因功能提供了可靠的工具。
This study aimed to utilize a mutant library of Mycobacterium tuberculosis variant(M.tuberculosis var.)bovis C68001 for high-throughput screening to identify key genes affecting biofilm formation.The mutant library of M.tuberculosis var.bovis C68001 was cultured in Sauton′s medium to select mutants with significant differences in biofilm phenotype.The location of mutated genes was determined by resistance marker rescue and sequencing,and the impact of these mutated genes on the antioxidant capacity and SDS resistance of the bacteria was assessed.Furthermore,a phage system was employed to construct strains with target gene deletion,complementation,and overexpression.The effects of the target genes on bacterial mycelium were investigated using scanning electron microscopy,and their influence on bacterial stress resistance and intracellular survival was evaluated by measuring the bacteria′s acid resistance,antioxidant capacity,SDS resistance,and survival within the J774A.1 mouse macrophage cell line.The study successfully screened eight biofilm-deficient strains from the mutant library,with four identified as single-insertion mutants.The mutated genes were Rv1096,Rv3425,Rv 3136,and Rv 3671c.Knockout,complementation,and overexpression strains of the Rv 3671c gene were constructed and verified by PCR and Western blot.These strains exhibited enhanced abilities in biofilm formation,SDS resistance,and intracellular survival.The Rv 3671c gene plays a crucial role in the biofilm formation and stress resistance of M.tuberculosis var.bovis C68001.The constructed mutant library of M.tuberculosis var.bovis C68001 provides an efficient tool for target gene screening and identification,offering a reliable approach for exploring the genetic functions of M.tuberculosis var.bovis C68001.
作者
时文健
徐磊
张泽
杨蕊
辛凌翔
王楠
陈祥
鑫婷
SHI Wenjian;XU Lei;ZHANG Ze;YANG Rui;XIN Lingxiang;WANG Nan;CHEN Xiang;XIN Ting(Key Laboratory of Animal Biosafety Risk Prevention and Control(North)of Ministry of Agriculture and Rural Affairs,Institute of Animal Sciences,Chinese Academy of Agricultural Sciences,Beijing 100193,China;College of Veterinary Medicine,Yangzhou University,Yangzhou 225009,China;China Institute of Veterinary Drug Control,Beijing 100081,China;College of Animal Science,Fujian Agriculture and Forestry University,Fuzhou 350002,China;Jiangsu Key Laboratory of Zoonosis,Yangzhou University,Yangzhou 225009,China)
出处
《畜牧兽医学报》
北大核心
2025年第8期3992-4006,共15页
ACTA VETERINARIA ET ZOOTECHNICA SINICA
基金
十四五重点研发计划(2024YFD1800301)
中央级公益性科研院所基本科研业务费专项课题(2022-YWF-ZYSQ-01)
江苏省人兽共患病学重点实验开放课题(R2211)。
