军团菌野生株与胞内分离株全菌蛋白比较  

Comparison of the total expressed protein of wild strain and intracellular strain of Legionella

作　　者：余泽英 韩蓉 陈建平[2] YU Zeying;HAN Rong;CHEN Jianping(Department of Pathogenic Biology,School of Basic Medical Science,Chengdu Medical College,Chengdu,Sichuan 610500,China;Department of Pathogenic Biology,West China School of Preclinical and Forensic Medicine,Sichuan University,Chengdu,Sichuan 610041,China)

机构地区：[1]成都医学院基础医学院病原生物学教研室,四川成都610500 [2]四川大学华西基础医学与法医学院病原生物系,四川成都610041

出　　处：《热带医学杂志》2024年第10期1384-1387,1399,F0004,共6页Journal of Tropical Medicine

基　　金：四川省科技厅项目(2022NSFC1679);成都医学院自然科学一般项目(19Z058)。

摘　　要：目的采用蛋白质组学方法,探索军团菌自然水体株与胞内分离株蛋白表达的差异。方法将菌株CD-5感染巨噬细胞16 h后裂解细胞分离军团菌,并命名为CD-5'。将CD-5和CD-5'分别用培养基培养后提取全菌蛋白,蛋白定量后通过SDS电泳和Western blot探索存在差异后,采用双向电泳联合MALDI-TOF-TOF质谱深入研究。结果CD-5全菌蛋白双向电泳图上获得214个蛋白点,CD-5'获得256个蛋白点,二者之间共有96个差异蛋白点。CD-5与CD-5'相比,CD-5有32个蛋白表达上调(编号:A01-A32),CD-5'有64个蛋白表达上调(编号:B01-B64)。对9个具有显著差异蛋白点进行酶解质谱分析,鉴定结果:A22为β-内酰胺酶,A23为苹果酸脱氢酶,A30为核苷酸绑定蛋白,A31为核糖体再循环因子,A32为假想蛋白,B03为30 s核糖体蛋白S1,B17为四型分泌系统蛋白Dot,B23为延伸因子-G片段,B62为烷基过氧化氢复原酶C亚基蛋白。结论胞内外军团菌蛋白表达具有差异,这些蛋白可能在军团菌生理、致病、毒力方面起到一定作用。Objective To analyze the total expressed proteins of wild strain and intracellular strain of Legionella,and lay a foundation for further understanding and study on Legionella.Methods The strain CD-5 of Legionella was used to infect macrophages for 16 hours,and then the cells were lysed to isolate the bacteria,which was named CD-5'.After culture in the medium,the whole bacterial protein was extracted.After protein quantification,SDS electrophoresis and Western blot were used to explore the differences.Two-dimensional electrophoresis combined with MALDI-TOF-TOF mass spectrometry was used for in-depth analysis.Results The preliminary data from SDS-PAGE and Western blot showed that the differentially expressed proteins exist between CD-5 and CD-5'.A total of 214 protein points were obtained on the twodimensional electrophoretogram of CD-5 complete bacterial protein,and 256 protein points were obtained on CD-5'.There were 96 different protein points between them.There were 32 up-regulated protein expressions in CD-5 and 64 up-regulated protein expressions in CD-5'.Among the differentially expressed protein spots,9 significantly different spots of them were identified and analyzed by mass spectrometry.A22 was beta-lactamase;A23 was a malate dehydrogenase(MDH);the A30 was the nucleotide-binding protein;A31 was a ribosome recycling factor(RRF);the A32 was the hypothetical protein;the B03 was 30 s ribosomal protein S1;the B17 was the typeⅣsecretion protein Dot;B23 was for the elongation factor Tu,partia(EF-T,partia);B62 was indicated as Alkyl hydroperoxide reductase subunit C-like protein.Conclusions There were differences between the total expressed protein of CD-5 and CD-5'.These proteins might play a certain role in the physiology,pathogenicity and virulence of Legionella.

关 键 词：军团菌 水体 胞内 全菌蛋白 

分 类 号：R517.7[医药卫生—内科学]