摘要
目的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1.0μg/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。
Objective 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 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 The optimal transfection dose(1.0μg/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 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.
作者
石姚佳
田湉
熊廷蓉
王于
张笑恺
邹全明
SHI Yaojia;TIAN Tian;XIONG Tingrong;WANG Yu;ZHANG Xiaokai;ZOU Quanming(School of Pharmacy,Henan University,Kaifeng,Henan Province,475004;Department of Microbiology and Biochemical Pharmacy,National Engineering Research Center of Immunological Products,Faculty of Pharmacy and Laboratory Medicine,Army Medical University(Third Military Medical University),Chongqing,400038;Department of Basic Courses,Noncommissioned Officers School,Army Medical University(Third Military Medical University),Shijiazhuang,Hebei Province,050081,China)
出处
《陆军军医大学学报》
CAS
CSCD
北大核心
2024年第8期815-821,共7页
Journal of Army Medical University
基金
国家自然科学基金面上项目(82201942)。
关键词
金黄色葡萄球菌
巨噬细胞
胞内感染
分泌蛋白
Staphylococcus aureus
macrophages
intracellular infection
secretory protein
