期刊
JOURNAL OF IMMUNOLOGICAL METHODS
卷 332, 期 1-2, 页码 129-141出版社
ELSEVIER SCIENCE BV
DOI: 10.1016/j.jim.2007.12.018
关键词
Anaplasma marginale; in vitro transcription and translation; IVTT; T-lymphocyte antigens; high-throughput screening
资金
- Intramural NIH HHS Funding Source: Medline
- NIAID NIH HHS [AI-053692, R01 AI053692] Funding Source: Medline
The ability to rapidly screen a complex pathogen proteome for proteins that elicit recall T-lymphocyte responses from immune individuals would accelerate vaccine development. An outer membrane fraction of the rickettsia] pathogen Anaplasma marginale induces protective immunity against infection and disease in cattle. We have used this immunization model to evaluate high-throughput screening of proteins expressed by in vitro transcription and translation (IVTT) for recognition by memory CD4(+) T-lymphocytes. Fifty selected vaccine candidate antigens identified from the A. marginale genome were expressed from transcriptionally active PCR products using an Escherichia coli-based IVTT system, and bead-affinity purified using antibodies to His and FLAG epitope tags. IVTT-expressed bead-bound antigens were processed and presented by antigen presenting cells to T-lymphocytes from outer membrane immunized animals and evaluated for immunogenicity in proliferation assays. Antigens that consistently stimulated responses were known T-cell antigens major surface protein (MSP)2, MSP3, VirB9, and VirB10 and newly identified T-cell antigens outer membrane protein (OMP)4, OMP9, elongation factor-Tu, Ana29, and OMA87. Specific T-cell stimulation was achieved even at low antigen concentration, and was highly sensitive when compared with unbound IVTT reaction products. This method allows rapid expression and identification of T-lymphocyte antigens for any pathogen for which the genome sequence is available. (C) 2008 Elsevier B.V. All rights reserved.
作者
我是这篇论文的作者
点击您的名字以认领此论文并将其添加到您的个人资料中。
推荐
暂无数据