Molecular Definition Of Filarial And Related Nonfilarial Genes And Proteins

丝虫及相关非丝虫基因和蛋白质的分子定义

• 批准号: 7592159
• 负责人: Thomas B Nutman
• 金额: $ 15.41万
• 依托单位: NATIONAL INSTITUTE OF ALLERGY AND INFECTIOUS DISEASES
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/7592159
• 关键词: Adult; Affinity; Antibodies; Baculoviruses; Binding; Brugia; Brugia malayi; CXC chemokine receptor 3; Diagnosis; Digestion; Drug Delivery Systems; Escherichia coli; Expressed Sequence Tags; Female; Filaria bancrofti; Gene Cluster; Gene Proteins; Genes; Genome; Ha antigen; Helminths; Human; IL5 gene; Immune response; Immunity; Immunization; Infection; Interleukin-5; Larva; Localized; Microfilaria; Molecular; Mus; Onchocerca volvulus; Open Reading Frames; PAWR protein; Parasites; Peptide Signal Sequences; Peptides; Phage Display; Proteins; Proteome; Proteomics; Publishing; Range; Serum; Shotguns; Signal Transduction; Staging; Strongyloides; Surface; Vaccines; Wolbachia; Yeasts; base; cDNA Library; eosinophil; immunogenic; interleukin-13 receptor; male; receptor; receptor coupling; research study

Using an EST (expressed sequence tag) approach, we have been able to identify greater than 965 gene clusters from Wuchereria bancrofti microfilariae based on1687 ESTs. Interestingly, a surprisingly high percentage (584/965; 60%) of these clustered ESTs contain a putative signal peptide, and only 6 appear to be GPI anchored. When compared to Brugia malayi (Bm) mf ESTs/Clusters, 45% of the Wb clusters had matches to Bm ESTs (identities ranging from 86-100%) whereas fewer (22%) matched Onchocerca volvulus (Ov) mf ESTs (with identities ranging from 80-100%). Using a phage display L3 cDNA library of Brugia malayi and screens with known human receptors, we have identified, cloned and characterized distinct molecules that bind to the human IL-5R, IL-10R, and IL-13R. The molecule we term, Bm-IL5RBP Brugia malayi IL5Rbinding protein has been expressed at high levels in a manner that retains its ability to bind to the human receptor. Antibodies raised to predicted immunogenic peptides inhibit the binding of rBm-IL5BP, and have been used to localize (by immuno-EM) Bm-IL5RBP to the surface of the infective stage larvae. The rBm-IL5BP does not itself prolong the survival of human eosinophils, but does inhibit the ability of human IL5 to prolong eosinophil survival. Interestingly, we have also identified a secreted product from Brugia malayi L3 that is chemotactic for human eosinophils. This secreted products signals through the G-coupled receptor CXCR3. As part of a consortium of many workers we have been part of the annotation of the filarial genome, a draft of which will be published in Sept, 2007. A separate proteomic analysis has also been completed using mass spectrospcopy in tandem with whole stage-specific tryptic digestion. Not only has the stage specific proteomes been completed for adult males, adult females, microfilariae, and infective larvae, but we have also completed the Brugia Wolbachia proteome and the excretory/secretory proteome. These analyses (still underway) have provided clear evidence that those genes encoding "hypothetical proteins" are real and have also identified close to 70% of all predicted open reading frames. An expanded approach to protective immunity in both filarial infections and in strongyloides is underway. ha Antigens affinity purified using sera from either mice immunized with irradiated larvae that show close to 100% protection to challenge infection with S. strongyloides or humans demonstrating immunity to Ss infection have identified 6 candidates that are being expressed in E. coli, yeast and baculovirus to determine which product can be made in large quantities to allow for protein based immunization experiments.

使用EST（表达的序列标签）方法，我们能够鉴定出基于1687 ESTS的Bancrofti微丝菌的965个基因簇。 有趣的是，这些聚类的EST中有一个令人惊讶的高百分比（584/965; 60％）包含推定的信号肽，并且似乎只有6个是GPI锚定的。与Brugia Malayi（BM）MF EST/集群相比，有45％的WB簇与BM EST的匹配（身份范围为86-100％），而在Chocerca volvulus（OV）MF ESTS上，较少的（22％）匹配的MF ESTS（与80-100％的身份差异）。 使用噬菌体显示的Brugia Malayi的L3 cDNA库和带有已知人体受体的筛选，我们已经鉴定出，克隆和表征与人IL-5R，IL-10R和IL-13R结合的不同分子。 我们称为BM-IL5RBP MALAYI IL5RBINDING蛋白的分子以高水平表达，以保持其与人体受体结合的能力。 提出的预测免疫原性肽的抗体抑制了RBM-IL5BP的结合，并已用于（通过免疫EM）将BM-IL5RBP定位于感染期幼虫表面。 RBM-IL5BP本身并不能延长人嗜酸性粒细胞的存活，而是抑制了人IL5延长嗜酸性粒细胞生存的能力。 有趣的是，我们还确定了Brugia Malayi L3的分泌产品，该产品对人嗜酸性粒细胞是趋化性的。该分泌的产品通过G偶联受体CXCR3发出信号。 作为许多工人财团的一部分，我们一直是丝状基因组注释的一部分，该草案将于2007年9月发布。 还使用质量光谱镜与整个阶段特异性的胰蛋白酶消化同时完成了单独的蛋白质组学分析。 不仅针对成年男性，成年女性，微毛虫和感染性幼虫完成了阶段的特异性蛋白质组织，而且我们还完成了Brugia Wolbachia蛋白质组和排泄/分泌蛋白质组。 这些分析（仍在进行中）提供了明确的证据，表明那些编码“假设蛋白质”的基因是真实的，并且还确定了近70％的预测开放式阅读框架的70％。 在丝状感染和强层中，正在扩大保护性免疫的方法。 HA抗原亲和力使用来自用辐照幼虫免疫的两只小鼠的血清纯化，这些血清表现出接近100％的保护，以挑战对SS SS感染免疫的人类或人类挑战感染，已经确定了6种候选物，这些候选者在大肠杆菌，酵母菌和西部酵母病毒中表达，以确定哪些产品可以允许蛋白质基于蛋白质化的实验。