Prokaryotic expression,purification,and in vivo and in vitro interaction studies of the potential virulence factors EsxA and EsxB of Mycobacterium haemophilum
10.3969/j.issn.1002-2694.2025.00.114
- VernacularTitle:嗜血分枝杆菌潜在毒力因子EsxA、EsxB的原核表达、纯化及体内外互作研究
- Author:
Rongxian XIE
1
;
Longyun CHENG
1
;
Xilu YUAN
1
;
Li LI
1
;
Haihong JIA
1
;
Bingqing LI
1
Author Information
1. 山东第一医科大学(山东省医学科学院)临床与基础医学院(基础医学研究所),济南 250062
- Publication Type:Journal Article
- Keywords:
Mycobacterium haemophilum;
EsxA;
EsxB;
protein extraction;
fluorescence colocalization
- From:
Chinese Journal of Zoonoses
2025;41(8):824-831
- CountryChina
- Language:Chinese
-
Abstract:
This studyobtained the proteins of virulence factors EsxA and EsxB from Mycobacterium haemophilum and explored their interactions both in vitro and in vivo.The aim was to lay a foundation for further analysis of the functional mechanisms of EsxA and EsxB,and to further the development of drugs targeting Mycobacterium haemophilum.On the basis of bioinformatics analysis of the virulence factors EsxA and EsxB from Mycobacterium haemophilum,we performed molecular cloning,using Mycobacterium haemophi-lum as a template to construct recombinant plasmids EsxA-pGl01,EsxB-pGl01,and EsxB-pET-29b.The proteins of EsxA,EsxB,and their complex were expressed with a prokaryotic system,then purified through nickel-affinity chromatography and gel filtration chromatography.Intracellular colocalization was determined through fluorescence colocalization.Prokaryotic expression systems for EsxA,EsxB,and their complex were successfully constructed,and purified proteins were obtained.Fluorescence colocalization indi-cated that EsxA and EsxB interacted in vivo.In vivo fluorescence colocalization and in vitro complex formation suggested that EsxA and EsxB interacted both intracellularly and extracellularly.Our findings lay a foundation for studying the pathogenic mechanisms of the virulence factors EsxA and EsxB in Mycobacterium haemophilum,and performing structural analysis of their complex.
