| 摘要: |
| 【目的】用绿色荧光标记的布鲁菌M5菌株（GFP-M5）侵染小鼠骨髓源性树突状细胞，对成熟树突状细胞表面主要组织相容性复合物Ⅱ（MHC Ⅱ）类分子结合的多肽进行筛选。【方法】用联合细胞因子重组小鼠GM-CSF和IL-4诱导小鼠骨髓单个核细胞向树突状细胞定向分化和增殖，于诱导培养的第1,3,5,7天在倒置荧光显微镜下观察细胞的形态学变化；用PE标记的CD11c抗体和FITC标记的CD80抗体对培养第7天的小鼠骨髓源性树突状细胞进行标记，流式细胞仪检测树突状细胞表面因子表达情况；用GFP-M5侵染培养第7天的小鼠骨髓源性树突状细胞24 h，激光共聚焦显微镜下观察菌株侵染情况；采用免疫共沉淀技术从树突状细胞膜水溶性蛋白溶液中分离纯化MHC Ⅱ-免疫多肽组复合物，C18固相萃取柱纯化小分子多肽，应用高效毛细管电泳(HPCE)和液相色谱-二级质谱（LC-MS/MS）联用技术进行纯化和测序，在布鲁菌基因组数据库中对质谱测试原始数据进行检索，筛选与布鲁菌M5基因组匹配的MHCⅡ类结合序列所代表的蛋白质。【结果】倒置荧光显微镜下观察细胞生长状态发现，小鼠骨髓源性树突状细胞由最初表面光滑无突起的圆形细胞（第1天）转变为表面突起明显增多的典型树突状细胞（第7天）；经流式细胞仪检测发现，培养第7天的树突状细胞CD11c阳性率为72.0%，CD80阳性率为8.4%，符合未成熟树突状细胞表面标志物的特征。GFP-M5侵染细胞24 h后在激光共聚焦显微镜下可观察到大部分树突状细胞已完全被侵染，即成为成熟树突状细胞；免疫共沉淀结合LC MS/MS技术，从经GFP-M5侵染的3组树突状细胞膜水溶性蛋白溶液中分离出了800多个MHC Ⅱ分子提呈的多肽分子，经与布鲁菌全基因组比对，属于布鲁菌且在3个试验重复中至少重复出现2次的有28个多肽序列；蛋白质鉴定表明，这些短肽包括布鲁菌外膜蛋白和外膜蛋白组装过程中需要的相关酶类，以及在抗原递呈过程中细胞内一系列生化反应所需的酶类。【结论】建立了外源性抗原体外侵染树突状细胞，获得了一系列布鲁菌M5菌株侵染小鼠骨髓源性树突状细胞后的MHC Ⅱ免疫多肽。 |
| 关键词: 树突状细胞 布鲁菌M5 组织相容性复合物Ⅱ 抗原肽 |
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| 基金项目:国家重点基础研究发展计划（973计划）项目“重要人兽共患胞内寄生菌病流行特征及病原致病机制研究”（2010CB-530200） |
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| Screening of antigen peptides after infection of mouse bone marrow-derived dendritic cells by Brucella M5 |
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ZHANG Fan,ZHOU Yongshun,CHEN Fulong,et al
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| Abstract: |
| 【Objective】Undulant M5 strain (GFP-M5) tagged by green fluorescent was used to infect mice bone marrow derived dendritic cells and peptides combined with major histocompatibility complex class Ⅱ(MHC Ⅱ) molecules on surface of mature dendritic cells were screened.【Method】Mice bone marrow mononuclear cells were induced to differentiate and proliferate into dendritic cells by recombinant mouse GM-CSF and IL-4 combined with cytokines.Morphological changes of cells were observed under inverted fluorescence microscope at days 1,3,5 and 7 of induction culture.PE labeled CD11c antibody and FITC labeled CD80 antibody were used to label bone marrow derived dendritic cells of mice on day 7 of culture,and the expression of surface factors of dendritic cells was detected by flow cytometry.GFP-M5 was used to infect bone marrow derived dendritic cells of mice on day 7 of culture for 24 h,and the infection was observed under laser confocal microscope.With immune co precipitation technique,MHC Ⅱ-immune polypeptide group complex was purified from water soluble protein solution of dendritic cell membrane.C18 column solid phase extraction column was used to purify small molecule polypeptides,and high performance capillary electrophoresis (HPCE) and liquid chromatography mass spectrometry (LC/MS/MS) was used for further purify and sequencing.Data retrieval from undulant genome database,and protein matching undulant M5 MHC genomes was used for retrieval.【Result】The growth of bone marrow cells was observed under inverted fluorescence microscope.The cells changed from smooth round cells with no prominences on surface (day 1) to typical dendritic cells with more prominences on surface (day 7).Flow cytometry showed that the positive rates of CD11c and CD80 were 72.0% and 8.4% on day 7,which were consistent with characteristics of surface markers of immature dendritic cells.Most GFP-M5 infected cells were completely infected under confocal laser microscope for 24 h,indicating that they were mature dendritic cells.The immune co precipitation with LC-MS/MS technology isolated more than 800 MHC Ⅱ peptide molecules from water soluble protein solution of dendritic cell membrane of GFP M5 infected groups.They were undulant based on genome wide comparison and there were 28 peptide sequences showing up at least two times among the three groups.Protein identification showed that these peptides included enzymes needed for the assembly of Brucella outer membrane proteins and outer membrane proteins,as well as for a series of biochemical reactions in cell during antigen presentation.【Conclusion】This study established the exogenous antigen afterin vitro infection of dendritic cells and obtained a series of MHC Ⅱ immune polypeptide groups after mice bone marrow derived dendritic cells were infected by undulant M5 strains. |
| Key words: dendritic cells Brucella M5 histocompatibility complex Ⅱ antigen peptide |
