两种处理方法对小鼠骨髓源性树突状细胞体外递呈布鲁氏菌免疫多肽组分析的影响  

Effects of Two Treatment on Analysis of Mouse Dendritic Cells forPresentation of Brucella abortus Immunopeptidome in vitro

作　　者：李婷婷 刘子超 周永顺 何营 耿方豪 陈洁然 高剑峰[1] LI Tingting;LIU Zichao;ZHOU Yongshun;HE Ying;GENG Fanghao;CHEN Jieran;GAO Jianfeng(College of Life Sciences,Shihezi University,Shihezi 832003,China)

机构地区：[1]石河子大学生命科学学院,石河子832003

出　　处：《中国畜牧兽医》2023年第9期3730-3739,共10页China Animal Husbandry & Veterinary Medicine

基　　金：国家重点基础研究发展计划(973计划)项目(2010CB530200)。

摘　　要：【目的】利用布鲁氏菌GFP-M5侵染小鼠骨髓源性树突细胞(DCs),比较侵染后细胞的两种处理方法对免疫多肽组分析的影响,建立布鲁氏菌侵染髓系DCs后免疫多肽组分离纯化技术。【方法】使用重组小鼠粒细胞巨噬细胞集落刺激因子(GM-CSF)与重组小鼠白介素4(IL-4)诱导培养小鼠骨髓细胞向DCs分化及增殖;然后用布鲁氏菌GFP-M5菌株侵染小鼠骨髓源性DCs后通过扫描电子显微镜观察侵染前后细胞表面形态变化。同时,利用两种方法处理布鲁氏菌侵染后的DCs:(1)试剂盒法:用Mem-PER^(TM)Plus试剂盒提取DCs表面抗原肽-主要组织相容性复合体Ⅱ(pMHCⅡ)复合物及其他膜蛋白组分,BCA法测定蛋白浓度;(2)低渗溶胀法:低渗缓冲液处理侵染后的细胞,计算不同离心力下的除菌率,通过倒置显微镜观察不同离心力下细胞膜碎片悬浮液中细胞膜的损耗程度,收集含pMCHⅡ复合物的细胞膜碎片悬浮液。通过免疫共沉淀(Co-IP)试验以及液相色谱-串联质谱联用技术(LC-MS/MS)分析,识别并筛选MHCⅡ结合的属于布鲁氏菌的肽段,并对两种处理方法获得的细胞膜蛋白组分中筛选出的肽段进行比较。【结果】扫描电镜观察用GFP-M5菌株侵染DCs后,细胞形态发生明显变化,附着在细胞表面的布鲁氏菌多集中在细胞表面的分支处,呈杆状。Mem-PER^(TM)Plus试剂盒提取2.5×10^(7)个细胞的膜蛋白,蛋白浓度为1.4918 mg/mL;共筛选出7个与MHCⅡ分子结合的布鲁氏菌的肽序列。在低渗缓冲液∶细胞悬液1∶20,3 000 r/min离心10 min的条件下,可以达到较高的除菌率(91.00%),同时细胞膜损耗较小;分离出的细胞膜共筛选出289个与MHCⅡ分子结合的布鲁氏菌的肽序列。【结论】试剂盒法处理所耗时间较短,后续试验处理较简便,但提取的膜蛋白样品中筛选出的MHCⅡ分子结合的肽序列较少;低渗溶胀法步骤比较简单易懂,所用试剂较少,处理的细胞膜碎片悬【Objective】Brucella abortus(B.abortus)GFP-M5 was used to infest mouse bone marrow-derived dendritic cells(DCs)to compare the effects of two treatments of the cells after infestation on immunopeptidome analysis and to establish a technique for the isolation and purification of immunopeptidome after B.abortus infestation of myeloid DCs.【Method】The differentiation and proliferation of mouse bone marrow cells to DCs were induced by recombinant mouse granulocyte macrophage colony-stimulating factor(GM-CSF)and recombinant mouse interleukin 4(IL-4).Then,mouse bone marrow-derived DCs were infected with B.abortus GFP-M5 strain and the morphological changes of cell surface before and after infection were observed by scanning electron microscopy.Meanwhile,two methods were used to treat DCs after B.abortus infestation:①Kit method:The antigenic peptide-major histocompatibility complexⅡ(PmhcⅡ)complex and other membrane protein components on the surface of DCs with Mem-PER ^(TM) Plus kit,and the protein concentration was determined by BCA method;②Hypotonic lysis method:The infested cells were treated with hypotonic buffer,and the sterilization rate was calculated under different centrifugal forces was calculated.The degree of cell membrane loss in the cell membrane debris suspension under different centrifugal forces was observed by inverted microscopy,and the cell membrane debris suspension containing the pMCHⅡcomplex was collected.Through co-immunoprecipitation(Co-IP)experiment and liquid chromatography tandem mass spectrometry(LC-MS/MS)analysis,MHCⅡ-bound peptides belonging to B.abortus were identified and screened,and the peptides screened from the cell membrane protein fractions obtained by the two methods were compared.【Result】Scanning electron microscopy showed that cell morphology changed significantly after DCs were infected with GFP-M5 strain.B.abortus attached to the cell surface were mostly concentrated at the branches of the cell surface,which were rod-shaped.Mem-PER ^(TM) Plus kit was used

关 键 词：布鲁氏菌 细胞膜蛋白 细胞膜分离 免疫多肽组 

分 类 号：S852.6[农业科学—基础兽医学] Q952.5[农业科学—兽医学]