基于结核分枝杆菌牛变种C68001随机突变体库筛选和鉴定成膜相关基因

doi:10.11843/j.issn.0366-6964.2025.08.037

畜牧兽医学报 ›› 2025, Vol. 56 ›› Issue (8): 3992-4006.doi: 10.11843/j.issn.0366-6964.2025.08.037

基于结核分枝杆菌牛变种C68001随机突变体库筛选和鉴定成膜相关基因

时文健¹^,²(), 徐磊³(), 张泽¹, 杨蕊¹^,⁴, 辛凌翔³, 王楠³, 陈祥⁵^,*(), 鑫婷¹^,*()

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), 北京 100193
2. 扬州大学兽医学院, 扬州 225009
3. 中国兽医药品监察所, 北京 100081
4. 福建农林大学动物科学学院, 福州 350002
5. 扬州大学江苏省人兽共患病学重点实验室, 扬州 225009

收稿日期:2025-01-03 出版日期:2025-08-23 发布日期:2025-08-28
通讯作者: 陈祥,鑫婷 E-mail:dz120230047@stu.yzu.edu.cn;609697832@qq.com;chenxiang@yzu.edu.cn;xinting@caas.cn
作者简介:时文健(1992-)，男，山东泰安人，博士生，主要从事牛结核病研究, E-mail: dz120230047@stu.yzu.edu.cn
徐磊(1983-)，男，山东日照人，副研究员，主要从事人畜共患病生物制品研究, E-mail: 609697832@qq.com
第一联系人：
时文健与徐磊为同等贡献作者
基金资助:
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)

Screening and Identification of Biofilm-related Genes Based on the Random Mutant Library of Mycobacterium tuberculosis Variant bovis C68001

SHI Wenjian¹^,²(), XU Lei³(), ZHANG Ze¹, YANG Rui¹^,⁴, XIN Lingxiang³, WANG Nan³, CHEN Xiang⁵^,*(), XIN Ting¹^,*()

1. 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
2. College of Veterinary Medicine, Yangzhou University, Yangzhou 225009, China
3. China Institute of Veterinary Drug Control, Beijing 100081, China
4. College of Animal Science, Fujian Agriculture and Forestry University, Fuzhou 350002, China
5. Jiangsu Key Laboratory of Zoonosis, Yangzhou University, Yangzhou 225009, China

Received:2025-01-03 Online:2025-08-23 Published:2025-08-28
Contact: CHEN Xiang, XIN Ting E-mail:dz120230047@stu.yzu.edu.cn;609697832@qq.com;chenxiang@yzu.edu.cn;xinting@caas.cn

摘要/Abstract

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、Rv3136和Rv3671c；构建Rv3671c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Rv3671c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。Rv3671c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。

关键词: 结核分枝杆菌牛变种, Himar 1转座子, 随机插入突变库, 生物膜, Rv3671c

Abstract:

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, Rv3136, and Rv3671c. Knockout, complementation, and overexpression strains of the Rv3671c gene were constructed and verified by PCR and Western blot. These strains exhibited enhanced abilities in biofilm formation, SDS resistance, and intracellular survival. The Rv3671c 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.

Key words: Mycobacterium tuberculosis variant bovis, Himar 1 transposon, mutant library, biofilm, Rv3671c

中图分类号:

S855.2

时文健, 徐磊, 张泽, 杨蕊, 辛凌翔, 王楠, 陈祥, 鑫婷. 基于结核分枝杆菌牛变种C68001随机突变体库筛选和鉴定成膜相关基因[J]. 畜牧兽医学报, 2025, 56(8): 3992-4006.

SHI Wenjian, XU Lei, ZHANG Ze, YANG Rui, XIN Lingxiang, WANG Nan, CHEN Xiang, XIN Ting. Screening and Identification of Biofilm-related Genes Based on the Random Mutant Library of Mycobacterium tuberculosis Variant bovis C68001[J]. Acta Veterinaria et Zootechnica Sinica, 2025, 56(8): 3992-4006.

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引物名称 Primer name	引物序列(5′→ 3′) Sequence	用途 Function
KanF	GATGGATTGCACGCAGGTTC	转座子插入鉴定
KanR	ATATCACGGGTAGCCAACGC
H1F	CGGGGACTTATCAGCCAACCTG	转座子插入区域侧翼扩增
H1R	TAGAGACCGGGGACTTATCAGCCAA
LFP	TTTTTTTT$\underline{GGCCTAAATGGCC}$CCTGCAAGCGGGAGCGACC	扩增Rv3671c基因左臂
LRP	TTTTTTTT$\underline{GGCCTTTCTGGCC}$CAGCATTGAGCCCAGCGCA
RFP	TTTTTTTT$\underline{CCATAGATTGG}$CAGGTGCTCGGTGTGGTGTTC	扩增Rv3671c基因右臂
RRP	TTTTTTTT$\underline{CCATCTTTTGG}$CGAGGCCGCTGGTCATACTG

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基于结核分枝杆菌牛变种C68001随机突变体库筛选和鉴定成膜相关基因

Screening and Identification of Biofilm-related Genes Based on the Random Mutant Library of Mycobacterium tuberculosis Variant bovis C68001

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