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.

利用EST（表达序列标签）的方法，我们已经能够确定超过965个基因簇的班氏丝虫基于1687 EST。 有趣的是，令人惊讶的是，这些成簇的EST中有很高的百分比（584/965; 60%）含有推定的信号肽，只有6个似乎是GPI锚定的。当与马来丝虫（Bm）mf EST/簇相比时，45%的Wb簇与Bm EST匹配（同一性范围为86-100%），而较少（22%）的Wb簇与盘尾丝虫（Ov）mf EST匹配（同一性范围为80-100%）。 利用噬菌体展示马来丝虫L3 cDNA文库和已知的人受体筛选，我们已经鉴定、克隆和表征了与人IL-5 R、IL-10 R和IL-13 R结合的不同分子。 我们称之为Bm-IL 5 RBP的分子马来丝虫IL 5 Rbinding蛋白以保持其与人受体结合能力的方式高水平表达。 针对预测的免疫原性肽产生的抗体抑制rBm-IL 5 BP的结合，并且已经用于将Bm-IL 5 RBP定位（通过免疫EM）到感染期幼虫的表面。rBm-IL 5 BP本身不延长人嗜酸性粒细胞的存活，但确实抑制人IL 5延长嗜酸性粒细胞存活的能力。 有趣的是，我们还鉴定了马来丝虫L3的分泌产物，其对人嗜酸性粒细胞具有趋化性。这种分泌产物通过G-偶联受体CXCR 3发出信号。 作为一个由许多工作者组成的联盟的一部分，我们一直是丝虫基因组注释的一部分，其草案将于2007年9月出版。 还使用质谱法与整个阶段特定的胰蛋白酶消化相结合完成了单独的蛋白质组学分析。 不仅完成了成年男性，成年女性，微丝蚴和感染性幼虫的阶段特异性蛋白质组，但我们也完成了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，证明那些编码“假设蛋白质”的基因是真实的，并且还鉴定了接近70%的所有预测开放阅读框架。 一种扩大的方法，以保护免疫的丝虫感染和类圆线虫正在进行中。 ha使用来自用辐射幼虫免疫的小鼠的血清亲和纯化的抗原，所述辐射幼虫对S.类圆线虫或对Ss感染表现出免疫力的人已经鉴定出6种在E.大肠杆菌，酵母和杆状病毒，以确定哪种产品可以大量生产，以允许基于蛋白质的免疫实验。