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金黄色葡萄球菌巨噬细胞胞内生存关键分泌蛋白筛选体系的构建
Construction of a screening system for key intracellular survival proteins of macrophages of
Staphylococcus aureus - 陆军军医大学学报 2024年46卷第8期 页码：815-821
- 作者机构：
1.475004 河南开封，河南大学药学院
2.400038 重庆，陆军军医大学(第三军医大学)药学与检验医学系微生物与生化药学教研室，国家免疫生物制品工程技术研究中心
3.050081 石家庄，陆军军医大学士官学校基础部
- 作者简介：
[邹全明，E-mail: qmzou2007@163.com ZOU Quanming, E-mail: qmzou2007@163.com
- 基金信息：国家自然科学基金(82201942)
DOI：10.16016/j.2097-0927.202311091
中图分类号： R341;R378.11;R392-33收稿：2023-11-09，
修回：2024-02-01，
纸质出版：2024-04-30
- 稿件说明：
移动端阅览
石姚佳, 田湉, 熊廷蓉, 等. 金黄色葡萄球菌巨噬细胞胞内生存关键分泌蛋白筛选体系的构建[J]. 陆军军医大学学报, 2024,46(8):815-821.
Yaojia SHI, Tian TIAN, Tingrong XIONG, et al. Construction of a screening system for key intracellular survival proteins of macrophages of
Staphylococcus aureus [J]. Journal of Army Medical University, 2024, 46(8): 815-821.石姚佳, 田湉, 熊廷蓉, 等. 金黄色葡萄球菌巨噬细胞胞内生存关键分泌蛋白筛选体系的构建[J]. 陆军军医大学学报, 2024,46(8):815-821. DOI： 10.16016/j.2097-0927.202311091.
Yaojia SHI, Tian TIAN, Tingrong XIONG, et al. Construction of a screening system for key intracellular survival proteins of macrophages of
Staphylococcus aureus [J]. Journal of Army Medical University, 2024, 46(8): 815-821. DOI： 10.16016/j.2097-0927.202311091.
摘要
目的
2
建立金黄色葡萄球菌(
Staphylococcus aureus
，
S. aureus
)巨噬细胞胞内感染状态下分泌蛋白高通量筛选体系，探索金葡菌胞内生存所需关键分泌蛋白。
方法
2
利用课题组建立的金葡菌分泌蛋白真核系统表达载体库，通过DNA转染表达技术，得到能够在细胞内表达金葡菌分泌蛋白的RAW264.7巨噬细胞阵列。金葡菌感染RAW264.7后，清除胞外菌，观察金葡菌胞内生存情况。最后通过构建相应分泌蛋白的过表达及敲除金葡菌验证筛选结果。
结果
2
通过探索RAW264.7质粒转染剂量、金葡菌感染复数(multiplicity of infection
MOI)以及感染时间，确立筛选体系的最优质粒转染剂量为1.0 μg/孔、MOI为1.0、感染时间为4 h。筛选结果结合相应分泌蛋白过表达和敲除菌株验证，成功筛选出hypothetical protein、Serine protease E等分泌蛋白具有促进胞内金葡菌生存功能。
结论
2
成功构建金葡菌巨噬细胞胞内生存关键分泌蛋白筛选体系。
Abstract
Objective
2
To establish a high-throughput screening system to obtain key
Staphylococcus aureus
(
S.aureus
) secretory proteins which required for
S.aureus
survival in macrophages.
Methods
2
Based on our validated eukaryotic expression vector library of
S.aureus
secretory proteins
DNA transfection was used to obtain an RAW264.7 macrophage array expressing
S.aureus
secretory proteins. After the RAW264.7 cells were infected with
S.aureus
the extracellular bacteria were removed to observe the intracellular surviving situation of
S.aureus
. Finally
the screening results were validated by the overexpression and knockout
S.aureus
of corresponding secretory proteins.
Results
2
The optimal transfection dose (1.0 μg/well) of plasmids for RAW264.7
multiplicity of infection (MOI
1.0)
and infection time (4 h after removing extracellular bacteria of
S.aureus
) were established respectively. To validate the screening results
the corresponding overexpression and knockout strains were constructed. And hypothetical protein and Serine protease E were found to promote the survival of intracellular
S.aureus
.
Conclusion
2
We successfully construct a screening system for key secreted secretory proteins which required for
S.aureus
surviving in macrophages
which may advance the study of the intracellular surviving mechanism of
S. aureus
.
关键词
Keywords
references
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