Identification of novel antigenic proteins in a complex Anaplasma marginale outer membrane immunogen by mass spectrometry and genomic mapping
Identification of novel antigenic proteins in a complex Anaplasma marginale outer membrane immunogen by mass spectrometry and genomic mapping
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DOI：
10.1128/iai.73.12.8109-8118.2005
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发表时间：
2005-12-01
影响因子：
3.1
通讯作者：
Brown, WC
中科院分区：
文献类型：
--
作者：
Lopez, JE;Siems, WF;Brown, WC
使用纯化的边缘无形体外膜进行免疫可诱导针对感染的完全保护，这与 CD4(+) T 淋巴细胞介导的 γ 干扰素分泌和免疫球蛋白 G2 (IgG2) 抗体滴度相关。然而，对外膜免疫原的组成的了解是有限的。最近对边缘曲霉基因组的测序和注释预测至少有 62 个外膜蛋白 (OMP)，从而能够使用蛋白质组和基因组方法通过使用外膜疫苗接种中的 IgG 血清抗体来鉴定新型 OMP。采用二维电泳分离外膜蛋白，免疫印迹法检测外膜接种牛免疫血清中总IgG和IgG2识别的蛋白。切除免疫反应蛋白点并进行液相色谱-串联质谱分析。对边缘曲霉基因组的数据库搜索鉴定出 24 种抗原蛋白，预计它们是外膜、内膜或膜相关蛋白。这些包括先前表征的表面暴露外膜蛋白 MSP2、操纵子相关基因 2 (OpAG2)、MSP3 和 MSP5 以及最近鉴定的附属物相关蛋白。在 21 种新描述的抗原蛋白中，有 14 种在边缘曲霉基因组中注释，包括 IV 型分泌系统蛋白、延伸因子 Tu 和 MSP2 超家族的成员。这些新型抗原蛋白的鉴定显着扩展了目前对保护性免疫原组成的理解，并为疫苗开发提供了新的候选者。
Immunization with purified Anaplasma marginale outer membranes induces complete protection against infection that is associated with CD4(+) T-lymphocyte-mediated gamma interferon secretion and immunoglobulin G2 (IgG2) antibody titers. However, knowledge of the composition of the outer membrane immunogen is limited. Recent sequencing and annotation of the A. marginale genome predicts at least 62 outer membrane proteins (OMP), enabling a proteomic and genomic approach for identification of novel OMP by use of IgG serum antibody from outer membrane vaccinates. Outer membrane proteins were separated by two-dimensional electrophoresis, and proteins recognized by total IgG and IgG2 in immune sera of outer membrane-vaccinated cattle were detected by immunoblotting. Immunoreactive protein spots were excised and subjected to liquid chromatography-tandem mass spectrometry. A database search of the A. marginale genome identified 24 antigenic proteins that were predicted to be outer membrane, inner membrane, or membrane-associated proteins. These included the previously characterized surface-exposed outer membrane proteins MSP2, operon associated gene 2 (OpAG2), MSP3, and MSP5 as well as recently identified appendage-associated proteins. Among the 21 newly described antigenic proteins, 14 are annotated in the A. marginale genome and include type IV secretion system proteins, elongation factor Tu, and members of the MSP2 superfamily. The identification of these novel antigenic proteins markedly expands current understanding of the composition of the protective immunogen and provides new candidates for vaccine development.