Characterization of the Lamprey Adaptive Immune System

七鳃鳗适应性免疫系统的表征

• 批准号: 7558272
• 负责人: Max Dale Cooper
• 金额: $ 38.01万
• 依托单位: EMORY UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2007
• 资助国家: 美国
• 起止时间: 2007-01-15 至 2011-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7558272
• 关键词: 2-Mercaptoethanol; Activated Lymphocyte; Affinity; Animals; Anthrax disease; Antibodies; Antibody Repertoire; Antigen Receptors; Antigens; Bacillus anthracis; Bacteria; Bacterial Model; Binding; Biochemical; Biological; C-terminal; Carbohydrates; Cell Line; Cells; Characteristics; Column Chromatography; Complementary DNA; Cystine; Data; Diagnostic tests; Disulfide Linkage; Drug or chemical Tissue Distribution; Electrons; Enzyme-Linked Immunosorbent Assay; Epitopes; Erythrocytes; Expression Library; Fc Receptor; Fishes; Fluorescence-Activated Cell Sorting; Fluorochrome; Generations; Genetic; Goals; Hagfish; Host Defense; Human; Immune response; Immune system; Immunity; Immunization; Individual; Jaw; Kinetics; Knowledge; Label; Lampreys; Larva; Learning; Leucine-Rich Repeat; Life; Ligand Binding; Link; Lymphocyte; Lymphocyte Activation; Mammalian Cell; Mammals; Mediating; Membrane; Memory; Methods; Microscopic; Mitogen Receptors; Modeling; Molecular; Mutation; Nature; Outcome; Particulate; Pathogenicity; Petromyzon marinus; Phospholipase; Production; Property; Proteins; Publishing; Recombinants; Research Personnel; Role; Salmonella typhimurium; Sampling; Sequence Analysis; Sorting - Cell Movement; Structure; Surface Plasmon Resonance; Testing; Time; Tissues; Vertebrates; antigen binding; cDNA Library; cellular transduction; design; disulfide bond; fast protein liquid chromatography; killings; leucine-rich repeat protein; pathogen; physical property; programs; protein complex; protein purification; receptor; receptor binding; receptor expression; research study; response

DESCRIPTION (provided by applicant): A second type of adaptive immune system has been discovered very recently in the extant jawless vertebrates. Lymphocytes in the lamprey and hagfish undergo somatic recombinatorial assembly of modular leucine-rich-repeat (LRR) genetic units to generate a potential variable lymphocyte receptor (VLR) repertoire estimated to be as large as the antibody repertoire in mammals; i.e., >1014. The antigen-specific VLRs that are generated in response to immunization could have many practical uses similar to those established for antibodies in a variety of diagnostic tests, protein purification, inhibition of intracellular proteins, pathogen identification, and pathogen inhibition. Toward the realization of this potential, the experiments in this proposal are designed to characterize functional and structural properties of the VLR-mediated adaptive immune system in the sea lamprey. The primary aims are to (i) define the immunization requirements, kinetics, and memory potential in the lamprey cellular and humoral immune responses to particulate antigens (Bacillus anthracis exosporium, Salmonella typhimurium, and human erythrocytes) and to soluble antigens (BcIA anthrax protein and its C-terminal domain), (ii) characterize the molecular nature of soluble VLRs in experiments designed to test the hypothesis that VLRs are released from individual lymphocytes as preformed multivalent protein complexes, (iii) identify individual antigen-specific VLR-bearing lymphocytes and isolate them in order to characterize the antigen-binding affinity and structure of their VLR products, and (iv) examine the potential of the VLR adaptive immune system for protective immunity against a model pathogen, S. typhimurium. These experiments involve VLR-bearing lymphocyte identification and isolation by fluorescence activated cell sorting for culture, electron microscopic analysis, generation of cDNA libraries, biochemical analysis of VLRs separated by chromatographic methods including affinity columns and fast protein liquid chromatography (FPLC), VLR cDNA sequencing and expression of recombinant VLR protein, mutation of VLR cystines in VLR transduced cell lines to examine their potential role in disulfide linkage to form the VLR multimers, and analysis of VLR antigen-binding affinity by surface plasmon resonance. In summary, it has recently been learned that jawless fish possess an adaptive immune system that features the production by their lymphocytes of a very large spectrum of antigen-specific receptors that differ structurally from our antibodies. Since these variable lymphocyte receptors (VLR) are as diverse as our antibodies and may have a relatively stable physical structure, they could prove very useful in detecting and inhibiting hazardous pathogens, like Bacillus anthracis. The experiments proposed here will define biological and structural features of the VLRs in order to explore their potential for health-related uses.

描述（由申请人提供）：最近在现存的无颌脊椎动物中发现了第二种类型的适应性免疫系统。七鳃鳗和盲鳗中的淋巴细胞经历模块化富含亮氨酸重复序列（LRR）遗传单元的体细胞重组组装，产生潜在的可变淋巴细胞受体（VLR）库，估计与哺乳动物中的抗体库一样大；即>1014。免疫反应产生的抗原特异性 VLR 可能具有许多实际用途，类似于在各种诊断测试、蛋白质纯化、细胞内蛋白质抑制、病原体识别和病原体抑制中为抗体建立的用途。为了实现这一潜力，本提案中的实验旨在表征海七鳃鳗中 VLR 介导的适应性免疫系统的功能和结构特性。主要目的是 (i) 确定七鳃鳗细胞和体液免疫反应中对颗粒抗原（炭疽杆菌外孢子、鼠伤寒沙门氏菌和人红细胞）和可溶性抗原（BciA 炭疽蛋白及其 C 末端结构域）的免疫要求、动力学和记忆潜力，(ii) 表征七鳃鳗的分子性质 在旨在测试 VLR 作为预先形成的多价蛋白复合物从单个淋巴细胞中释放的假设的实验中，研究可溶性 VLR，(iii) 识别单个抗原特异性 VLR 承载淋巴细胞并分离它们，以表征其 VLR 产物的抗原结合亲和力和结构，以及 (iv) 检查 VLR 适应性免疫系统的潜力 针对模型病原体鼠伤寒沙门氏菌的保护性免疫。这些实验包括通过荧光激活细胞分选来鉴定和分离携带VLR的淋巴细胞进行培养、电子显微镜分析、cDNA文库的生成、通过色谱方法（包括亲和柱和快速蛋白液相色谱（FPLC））分离的VLR的生化分析、VLR cDNA测序和重组VLR蛋白的表达、VLR胱氨酸的突变 在VLR转导的细胞系中检查它们在二硫键连接形成VLR多聚体中的潜在作用，并通过表面等离子体共振分析VLR抗原结合亲和力。总之，最近了解到，无颌鱼拥有适应性免疫系统，其特点是淋巴细胞产生大量的抗原特异性受体，这些受体在结构上与我们的抗体不同。由于这些可变淋巴细胞受体（VLR）与我们的抗体一样多样化，并且可能具有相对稳定的物理结构，因此它们在检测和抑制炭疽杆菌等有害病原体方面非常有用。这里提出的实验将定义 VLR 的生物学和结构特征，以探索其在健康相关用途中的潜力。