基于Antares2的金黄色葡萄球菌生物发光示踪系统的构建

胡珍; 尚伟龙; 饶贤才

doi:10.16016/j.2097-0927.202212145

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基于Antares2的金黄色葡萄球菌生物发光示踪系统的构建

基础医学 | 更新时间：2026-01-09

- 基于Antares2的金黄色葡萄球菌生物发光示踪系统的构建
- Construction of Staphylococcus aureus bioluminescence tracing system based on Antares2
- 陆军军医大学学报 2023年45卷第5期页码：393-399
- 作者机构：
  
  400038 重庆，陆军军医大学(第三军医大学)基础医学院微生物学教研室
- 作者简介：
  
  [饶贤才，E-mail: raoxiancai@126.com RAO Xiancai, E-mail: raoxiancai@126.com
- 基金信息：
  
  国家自然科学基金面上项目(82272341)
- DOI：10.16016/j.2097-0927.202212145
  中图分类号： R378.11;R394-33;R446.5
- 收稿：2022-12-27，
  
  修回：2023-01-17，
  
  纸质出版：2023-03-15
- 稿件说明：
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胡珍, 尚伟龙, 饶贤才. 基于Antares2的金黄色葡萄球菌生物发光示踪系统的构建[J]. 陆军军医大学学报, 2023,45(5):393-399.

Zhen HU, Weilong SHANG, Xiancai RAO. Construction of Staphylococcus aureus bioluminescence tracing system based on Antares2[J]. Journal of Army Medical University, 2023, 45(5): 393-399.
胡珍, 尚伟龙, 饶贤才. 基于Antares2的金黄色葡萄球菌生物发光示踪系统的构建[J]. 陆军军医大学学报, 2023,45(5):393-399. DOI： 10.16016/j.2097-0927.202212145.

Zhen HU, Weilong SHANG, Xiancai RAO. Construction of Staphylococcus aureus bioluminescence tracing system based on Antares2[J]. Journal of Army Medical University, 2023, 45(5): 393-399. DOI： 10.16016/j.2097-0927.202212145.

摘要

目的

构建融合型荧光素酶标记的金黄色葡萄球菌示踪菌株，建立金葡菌示踪系统。

方法

采用同源重组技术将融合型荧光素酶Antares2编码基因敲入到金葡菌USA300基因组中，与金葡菌烯醇酶编码基因

eno

融合，构建稳定表达的示踪菌株。将示踪菌株与不同的底物混合，检测生物发光强度差异，筛选合适的菌株/底物示踪系统，并探究其在体外和体内的敏感性。

结果

通过构建基因敲入质粒pBT2-

eno-antares

2，转化金葡菌USA300菌株中进行基因敲入菌株筛选，经DNA测序和Western blot鉴定示踪菌株USA300/Eno-Antares2构建成功。细菌生长曲线和溶血实验表明所构建的示踪菌株与野生型USA300无明显差异。将示踪菌与DTZ、FUR和HFZ底物混合，肉眼均可观察到发光。不同浓度不同底物分析显示，USA300/Eno-Antares2/HFZ组成的系统发光性能最优，体外发光示踪的敏感性为50 CFU(菌落形成单位)，小鼠体内示踪敏感性为100 CFU。

结论

金葡菌USA300/Eno-Antares2/HFZ示踪系统性能佳，可用于金葡菌感染、定植、播散等研究。

Abstract

Objective

To construct a new fusion luciferase-labeled

Staphylococcus aureus

(

S. aureus

) tracing system.

Methods

S. aureus

tracer strain was constructed by using DNA homologous recombination technique. The gene encoding fusion luciferase Antares2 was knocked in the genome of

S. aureus

strain USA300 by fusing with the enolase (Eno) encoding gene. The bioluminescence intensity was analyzed by mixing with the tracer strain and different substrates. The suitable tracer/substrate system was selected based on the detection of bioluminescence intensity. The sensitivity of the tracer/substrate system

in vitro

and

in vivo

was also evaluated.

Results

The knock-in plasmid pBT2-

eno-antares

2 was constructed. After transformed into

S. aureus

USA300

the Antares2 knock-in strain was screened out. The tracer strain USA300/Eno-Antares2 was identified by DNA sequencing as well as Western blot detection of

Eno-Antares2 fusion proteins. Bacterial growth curve and hemolytic activity tests showed comparable results between USA300/Eno-Antares2 tracer strain and its wild-type USA300. USA300/Eno-Antares2 catalyzed substrates diphenylterazine (DTZ)

furimazine (FUR)

and hydrofurimazine (HFZ) to produce macroscopical bioluminescence. Analysis of different substrates at diverse concentrations revealed that USA300/Eno-Antares2/HFZ system exhibited the best performance. This system enabled reliable bioluminescence tracking of

S. aureus

with sensitivities of approximately 50 colony forming unit (CFU)

in vitro

and 100 CFU

in vivo

Conclusion

S. aureus

USA300/Eno-Antares2/HFZ tracing system is a good tool

which can be used for the study of

S. aureus

infection

colonization

and dissemination.

关键词

Keywords

references

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