关键词: 布鲁氏菌; 细胞膜蛋白; 细胞膜分离; 免疫多肽组

Abstract: 【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:① Kit method:The antigenic peptide-major histocompatibility complex Ⅱ(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;② Hypotonic 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 pMCH Ⅱ complex was collected.Through co-immunoprecipitation(Co-IP) experiment and liquid chromatography tandem mass spectrometry(LC-MS/MS) analysis,MHC Ⅱ-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 to extract membrane proteins from 2.5×10⁷ cells,the protein concentration was 1.4918 mg/mL.A total of 7 peptide sequences of B. abortus that bind to MHC Ⅱ molecules were screened.Under the conditions of hypotonic buffer:Cell suspension 1:20 and centrifugation at 3 000 r/min for 10 min,a high removal rate (91.00%) could be achieved with low loss of cell membranes.289 peptide sequences of B. abortus that bound to MHC Ⅱ molecules were screened from the isolated cell membranes.【Conclusion】 The reagent cassette method took less time to process and was easier to handle in subsequent experiments,but fewer MHC Ⅱ molecule-bound peptide sequences were screened from the extracted membrane protein samples.The hypotonic lysis method had simpler and easier to understand steps,used fewer reagents and more MHC Ⅱ molecule-bound peptide sequences were screened from the treated cell membrane fragment suspensions,but the treatment process resulted in the loss of some pMHC complexes.This experiment provided the basis for the development of a new vaccine for B.abortus.

Key words: Brucella abortus; cell membrane proteins; cell membrane isolation; immunopeptidome