期刊
JOURNAL OF IMMUNOLOGICAL METHODS
卷 373, 期 1-2, 页码 111-126出版社
ELSEVIER
DOI: 10.1016/j.jim.2011.08.007
关键词
T cell epitope discovery; Protein expression library; Altered peptide ligands; Prostate specific antigen; Francisella tularensis; High-throughput screening
资金
- National Institutes of Health [NO1-AI-40086, R01-AI-078345]
- NIH/NIAID Southeast Regional Center of Excellence for Emerging Infections and Biodefense (SERCEB) [U54 AI 057157]
- NIAID [T32-AI-007362, T32-AI-007285]
- NIH/NIAID [AI-073785]
Epitopes are a hallmark of the antigen specific immune response. The identification and characterization of epitopes is essential for modern immunologic studies, from investigating cellular responses against tumors to understanding host/pathogen interactions especially in the case of bacteria with intracellular residence. Here, we have utilized a novel approach to identify T cell epitopes exploiting the exquisite ability of particulate antigens, in the form of beads, to deliver exogenous antigen to both MHC class I and class II pathways for presentation to T cell hybridomas. In the current study, we coupled this functional assay with two distinct protein expression libraries to develop a methodology for the characterization of T cell epitopes. One set of expression libraries containing single amino acid substitutions in a defined epitope sequence was interrogated to identify epitopes with enhanced T cell stimulation for a MHC class I epitope. The second expression library is comprised of the majority of open reading frames from the intracellular pathogen and potential biowarfare agent, Francisella tularensis. By automating aspects of this technology, we have been able to functionally screen and identify novel T cell epitopes within F. tularensis. We have also expanded upon these studies to generate a novel expression vector that enables immunization of recombinant protein into mice, which has been utilized to facilitate T cell epitope discovery for proteins that are critically linked to Francisella pathogenicity. This methodology should be applicable to a variety of systems and other pathogens. (C) 2011 Elsevier B.V. All rights reserved.
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