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

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4 093个基因。成功筛选到温敏型噬菌体，并且滴度高于1×10¹¹ PFU/mL，以感染复数(multiplicity of infection, MOI)为10侵染菌株C68001 3 h后获得9 216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。

关键词:结核分枝杆菌牛变种;MycoMar T7;突变体库;噬菌体

Abstract:

[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 4 093 genes. A temperature-sensitive phage with a titer higher than 1×10¹¹ PFU/mL was successfully screened. After infecting C68001 at MOI=10 for 3 h, 9 216 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.

Key words:Mycobacterium tuberculosis variant bovis;MycoMar T7;mutant library;phage