结核分枝杆菌牛变种C68001转座子突变体库的构建  

Construction of an insertional mutant library of Mycobacterium tuberculosis variant bovis

作　　者：时文健 徐磊[3] 张泽 杨蕊 张广智 许霄峰 辛凌翔[3] 王楠[3] 鑫婷[1] SHI Wenjian;XU Lei;ZHANG Ze;YANG Rui;ZHANG Guangzhi;XU Xiaofeng;XIN Lingxiang;WANG Nan;XIN Ting(Key Laboratory of Animal Biosafety Risk Prevention and Control(North)&Key Laboratory of Veterinary Biological Products and Chemical Drugs of MARA,Institute of Animal Sciences,Chinese Academy of Agricultural Sciences,Beijing 100193,China;College of Veterinary Medicine,Yangzhou University,Yangzhou 225009,Jiangsu,China;China Institute of Veterinary Drug Control,Beijing 100081,China)

机构地区：[1]中国农业科学院北京畜牧兽医研究所,农业农村部动物生物安全风险预警及防控重点实验室(北方)农业农村部兽用生物制品与化学药品重点实验室,北京100193 [2]扬州大学兽医学院,江苏扬州225009 [3]中国兽医药品监察所,北京100081

出　　处：《微生物学通报》2025年第2期703-712,共10页Microbiology China

基　　金：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)。

摘　　要：【背景】结核分枝杆菌牛变种(Mycobacterium tuberculosis variant bovis)可以感染人和多种动物,是重要的人兽共患病原菌。【目的】以结核分枝杆菌牛变种C68001为母本菌株,利用MycoMar T7转座子系统,构建结核分枝杆菌牛变种突变体库,为深入探究结核分枝杆菌牛变种基因功能和致病机制提供技术平台。【方法】以C68001为母本菌株,对其全基因序列进行分析预测;筛选温敏型噬菌体,优化噬菌体扩增条件和侵染条件;获得高库容的结核分枝杆菌牛变种C68001突变体库。【结果】基于Nanopore三代和二代测序技术绘制细菌基因组完成图,C68001的完整基因组大小为4.342 Mb,含有4093个基因。成功筛选到温敏型噬菌体,并且滴度高于1×10^(11 )PFU/mL,以感染复数(multiplicity of infection,MOI)为10侵染菌株C680013 h后获得9216个单克隆的转座子突变体,随机挑选7个克隆进行PCR鉴定,证实转座子成功插入母本菌株。【结论】成功构建了结核分枝杆菌牛变种C68001的突变体库。[Background]Mycobacterium tuberculosis variant bovis is a zoonotic pathogen capable of causing tuberculosis in both humans and animals.[Objective]To construct a mutant library of M.tuberculosis var.bovis with M.tuberculosis var.bovis C68001 as the maternal strain and the MycoMar T7 transposon system,providing a technical platform for further investigation of the gene functions and pathogenic mechanisms of M.tuberculosis var.bovis.[Methods]The whole genome sequence of M.tuberculosis var.bovis C68001 was systematically analyzed.The temperature-sensitive phages were screened and the conditions of phage proliferation and infection were optimized.Finally,a mutant library with high storage capacity for M.tuberculosis var.bovis C68001 was obtained.[Results]Based on Nanopore third-generation and second-generation sequencing technology,the whole genome of C68001 was 4.342 Mb in size and contained 4093 genes.A temperature-sensitive phage with a titer higher than 1×10^(11) PFU/mL was successfully screened.After infecting C68001 at MOI=10 for 3 h,9216 monoclonal transposon mutants were obtained.Seven clones were randomly selected for PCR,which confirmed that transposons were successfully inserted into C68001.[Conclusion]The mutant library of M.tuberculosis var.bovis C68001 was successfully constructed.

关 键 词：结核分枝杆菌牛变种 MycoMar T7 突变体库 噬菌体 

分 类 号：S852.61[农业科学—基础兽医学]