鼠伤寒沙门菌六型分泌系统效应蛋白Clpv对巨噬细胞极化的影响  

Effect of T6SS effector protein Clpv of Salmonella typhimurium on macrophage polarization

作　　者：杜付熙 王文婕 尚珂[1,2,3] 余祖华[1,2,3] 李静[1,2,3] 贾艳艳[1,2,3] 廖成水[1,2,3] 丁轲[1,2,3] 张春杰[1,2,3] 程相朝[1,2,3] 陈松彪[1,2,3] DU Fuxi;WANG Wenjie;SHANG Ke;YU Zuhua;LI Jing;JIA Yanyan;LIAO Chengshui;DING Ke;ZHANG Chunjie;CHENG Xiangchao;CHEN Songbiao(Luoyang Key Laboratory of Live Carrier Biomaterial and Animal Disease Prevention and Control,Henan University of Science and Technology,Luoyang 471003,Henan,China;Laboratory of Functional Microbiology and Animal Health,Henan University of Science and Technology,Luoyang 471003,Henan,China;College of Animal Science and Technology,Henan University of Science and Technology,Luoyang 471003,Henan,China)

机构地区：[1]河南科技大学、洛阳市活载体生物材料与动物疫病防控重点实验室,河南洛阳471003 [2]河南科技大学功能微生物与畜禽健康实验室,河南洛阳471003 [3]河南科技大学动物科技学院,河南洛阳471003

出　　处：《微生物学报》2023年第7期2620-2632,共13页Acta Microbiologica Sinica

基　　金：河南省自然科学基金(232300421263);国家自然科学基金(31572489);河南科技大学博士研究启动专项基金(13480104)。

摘　　要：【目的】探讨Ⅵ型分泌系统(typeⅥsecretion system,T6SS)效应蛋白Clpv在鼠伤寒沙门菌(Salmonella enterica serovar Typhimurium)致病过程中的功能。【方法】以鼠伤寒沙门菌SL1344基因组为模板克隆clpv基因,并比较与其他革兰氏阴性菌台湾假单胞菌(Pseudomonas taiwanensis)、植生拉乌尔菌(Raoultella planticola)、鳗利斯顿氏菌(Listonella anguillarum)、菠萝多源菌(Pantoea ananatis)、粘放线菌(Actinomyces viscosus)和大肠埃希菌(Escherichia coli)的同源性;将clpv基因克隆至pEGFP-N1载体构建重组质粒pEGFP-Clpv,利用Western blotting、实时荧光定量聚合酶链式反应(real-time quantitative polymerase chain reaction,q-PCR)、荧光显微镜以及流式细胞术检测蛋白表达、定位及诱导小鼠巨噬细胞M1型和M2型极化水平。【结果】clpv基因全长为2637 bp,与台湾假单胞菌的同源性最高;Western blotting、qPCR和免疫荧光检测表明重组蛋白大小约120 kDa,在细胞中有明显绿色荧光并且主要定位于细胞膜;q-PCR和流式细胞术结果发现Clpv转染组巨噬细胞M1型极化显著增加(P<0.01),M2型巨噬细胞极化显著减少(P<0.01)。【结论】成功克隆表达鼠伤寒沙门菌T6SS效应蛋白Clpv,并明确其胞内表达定位以及对巨噬细胞极化的影响。[Objective]To investigate the role of type VI secretion system(T6SS)effector protein Clpv in the pathogenesis of Salmonella typhimurium.[Methods]We cloned the clpv gene from SL1344 genome of S.typhimurium and compared it with the clpv genes of other Gram-negative bacteria(Pseudomonas taiwanensis,Raoultella planticola,Listonella anguillarum,Pantoea ananatis,Actinomyces viscosus,and Escherichia coli).The clpv gene was cloned into pEGFP-N1 vector to construct the recombinant plasmid pEGFP-Clpv.The protein expression,localization,and the induced M1 and M2 polarization of mouse macrophages were detected by Western blotting,q-PCR,fluorescence microscopy,and flow cytometry.[Results]The clpv gene was 2637 bp,showing the highest homology to clpv of P.taiwanensis.Western blotting,q-PCR,and immunofluorescence showed that the recombinant protein was about 120 kDa and apparent green fluorescence in cells was found.The protein was located primarily in cell membrane.The results of q-PCR and flow cytometry indicated that Clpv enhanced M1 polarization(P<0.01)but weakened M2 polarization of mouse macrophages(P<0.01).[Conclusion]The T6SS effector protein Clpv of S.typhimurium was cloned and expressed,and its intracellular localization and effect on macrophage polarization were clarified.

关 键 词：鼠伤寒沙门菌 Ⅵ型分泌系统 Clpv蛋白 表达定位 极化 

分 类 号：R392[医药卫生—免疫学]