DEVELOPMENT OF THE ANTIBODY REPERTOIRE

抗体库的开发

• 批准号: 6691661
• 负责人: Harry William Schroeder
• 金额: $ 28.7万
• 依托单位: UNIVERSITY OF ALABAMA AT BIRMINGHAM
• 依托单位国家: 美国
• 财政年份: 2001
• 资助国家: 美国
• 起止时间: 2001-01-15 至 2005-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6691661
• 关键词: B lymphocyte; Orthomyxoviridae; Streptococcus pneumoniae; antibody formation; antibody specificity; autoantibody; autoimmune disorder; cell differentiation; gene expression; gene targeting; immunoglobulin genes; laboratory mouse

DESCRIPTION (investigator's abstract): The capacity to distinguish self from non-self depends on the ability to generate a diverse, yet balanced, repertoire of antigen-binding receptors. The mechanisms that enhance, as well as those that constrain, diversity are thus of fundamental interest. The primary aim of this study is to define the constraints imposed upon the third complementarity determining region of the immunoglobulin heavy chain (HCDR3). HCDR3 lies at the center of the antigen-binding site and typically determines antibody specificity. At the genetic level, HCDR3 represents the focal point of diversification of the pre-immune B-cell repertoire since it is the direct product of V(D)J recombination. The DH (diversity) gene segments compose the core of HCDR3 and are potentially translatable in six reading frames, each with a characteristic hydropathicity - charged, hydrophobic, or hydrophilic. The sequence composition of DH gene segments is conserved from shark to mouse to man, and diverse mechanisms are used in these species to ensure that the final HCDR3 repertoire is enriched for a neutral, hydrophilic sequence. Utilization of charged or hydrophobic HCDR3 domains is common only in exceptional circumstances (e.g., pathogenic anti-DNA autoantibodies). The hypothesis to be tested holds that use of alternative reading frames (or hydropathicities) is deleterious due either to production of unstable H chains, to reduced generation of antibodies of beneficial specificity, or to formation of antibodies with improper specificities (e.g., autoantibodies). Cre-loxP gene targeting will be used to generate mice wherein the antibody repertoire has been globally altered to produce HCDR3 intervals encoded by alternative DH reading frames. The DH locus will be reduced to a single, frame-shifted DH gene segment. These alterations will prejudice lymphotype precursors to produce germline-encoded charged or hydrophobic HCDR3s intervals, while maintaining a normal developmental pattern of V(D)J rearrangement. The effect of this redirection on the neonatal and adult response to antigen, and on the predisposition of these mice to diseases of altered immune function, will be examined. B-cell development will be monitored in neonatal and adult mice, and in adult TdT-/- mice that lack the ability to adjust hydropathicity by N-region addition. As a functional measure of the capacity to respond normally to antigen, mutant mice will be challenged with T-independent and T-dependent antigens, and with Strep. pneumoniae and influenza virus, to determine whether alteration of the repertoire can prove lethal to the organism. Finally, the effect that alteration of HCDR3 has on the development of the natural autoantibody repertoire and on the progression of autoimmune disease in the MRL-lpr/lpr mouse will be examined.

描述（研究者的摘要）：区分自我与 非自我取决于产生多样化但平衡的曲目的能力 抗原结合受体。增强的机制以及 因此，多样性是基本的利益。主要目的 这项研究是为了定义对第三个互补性施加的约束 确定免疫球蛋白重链（HCDR3）的区域。 HCDR3位于 抗原结合位点的中心，通常确定抗体 特异性。在遗传水平，HCDR3代表 免疫前B细胞曲目的多样化，因为它是直接的 V（d）J重组的产物。 DH（多样性）基因段组成 HCDR3的核心，并有可能在六个读取帧中翻译 一种特征性的水性质 - 充电，疏水或亲水性。这 DH基因段的序列组成从鲨鱼到小鼠保守 人类和各种机制用于这些物种，以确保最终 HCDR3曲目富含中性的亲水性序列。利用率 充电或疏水性HCDR3域仅在特殊 情况（例如致病性抗DNA自身抗体）。假设是 经过测试的使用替代阅读框（或水质疗法）是 由于生产不稳定的H链，有害 产生有益特异性的抗体，或形成 具有不当特异性（例如自身抗体）的抗体。 Cre-loxp基因 靶向将用于生成抗体库有的小鼠 在全球范围内改变以产生由替代DH编码的HCDR3间隔 阅读框架。 DH基因座将减少为单个瞬变的DH基因 部分。这些改变将损害产生的淋巴型前体 生殖线编码的电荷或疏水HCDR3S间隔，同时保持 V（d）J重新排列的正常发育模式。这个效果 对新生儿和成人对抗原的反应的重定向，以及 这些小鼠对免疫功能改变的疾病的易感性将是 检查。 B细胞的发育将在新生儿和成年小鼠中进行监测，以及 在成年TDT - / - 小鼠中缺乏通过N区调节氢化性的能力 添加。作为正常反应能力的功能衡量 抗原，突变小鼠将受到独立和T依赖性的挑战 抗原和链球菌。肺炎和流感病毒，以确定是否是否 曲目的改变可以证明有机体致命。最后， HCDR3改变对自然发展的影响 自身抗体曲目和自身免疫性疾病的进展 将检查MRL-LPR/LPR鼠标。