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: 6098951
• 负责人: Thomas B Nutman
• 金额: --
• 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/6098951
• 关键词: Filarioidae; Nematoda; Wuchereria; clinical research; enzyme linked immunosorbent assay; epitope mapping; filariasis; genetic library; helminthic antigen; human subject; immunity; microorganism genetics; molecular genetics; nucleic acid sequence; species difference

Using sera from individuals identified as "putatively immune" to Onchocerca volvulus infections, cDNA expression libraries have been screened; approximately 60 L3 and L4 recombinants have been identified and gridded for counter-screening with sera from well- defined patients with early patent infection. Concurrently, each has had partial sequence obtained (EST analysis and the recombinants have been placed in groups based on their homologies to known proteins in the databases although many have been shown to encode novel proteins). Genes encoding the four most promising (as vaccine targets) have been placed in expression vectors (yeast, bacterial-based expression systems) and the recombinant proteins are being characterized. This includes a filarial 1,6 bis phosphate aldolase that not only induces a strong immune response in animals, but also is a target of T cells in putatively immune individuals. Identifying and characterizing those molecules from the larval stages of the filarial parasites that induce protective immunity in lymphatic filariasis has also remained a large part of our effort. Using novel T-cell-based and antibody-based screening techniques, we have identified 12 vaccine targets from Wuchereria bancrofti larval cDNA expression libraries. The approach will be both traditional (over-expression of recombinant products) and DNA-based using both the proprietary Vical vectors as well as several re-engineered vectors that have shown promise in other systems. The development of diagnostic antigens for lymphatic filariasis has also been undertaken. To this end, a cDNA expression library from adult female parasites was constructed and differentially screened with appropriate sera. More than 25 individual recombinants have been identified as potentially diagnostic, and expression and extensive testing with large panels of well- defined sera has identified four recombinants that appeared to be specific for individuals with lymphatic filariasis. Sequence analysis indicated that 3/4 were interrelated and homologous to HMG-like proteins; one was unique and not homologous to any protein in the various databases. The unique polypeptide, termed Wb1.2 and one of the HMG-like proteins, Wb5.4, were expressed at high levels in several different prokaryotic expression vectors. Partially purified protein was then used as the basis for immunoblot assays using well-characterized sera from individuals with W. bancrofti infection (n=36) and B. malayi infection (n=9) as well as from individuals with nonlymphatic filarial infections (n=27), and nonfilarial parasitic infections (n=36). Normal individuals without parasites (n=15) were also studied. Using these recombinants alone or in combination gave sensitivities that ranged from 65 to 98% and specificities from 89 to 100%. In addition, sets of overlapping peptides have been synthesized and utilized to map the B-cell epitopes of these two proteins in the hopes of utilizing a peptide-based assay for diagnosis of patent filarial infection.

使用来自被鉴定为“脓毒症”的个体的血清 盘尾丝虫感染的“免疫”，cDNA表达 已筛选了约60个L3和L4文库 重组体已经被鉴定并网格化， 用来自明确定义的早期乳腺癌患者的血清进行反筛选 明显感染同时，每一个都有部分序列 获得（EST分析和重组体已被放置在 基于它们与已知蛋白质的同源性， 尽管许多数据库已经显示出编码新颖的 蛋白质）。编码四种最有希望的基因（作为疫苗） 靶）已被置于表达载体（酵母， 基于细菌的表达系统）和重组蛋白 正在被定性。这包括一个丝虫1，6双磷酸盐 醛缩酶不仅在动物中诱导强烈的免疫应答， 也是免疫个体中T细胞的靶点。 从幼虫身上识别和鉴定这些分子 诱导保护性免疫的丝虫阶段， 淋巴丝虫病也仍然是我们努力的一大部分。 使用新的基于T细胞和基于抗体的筛选技术， 我们已经从班氏吴策线虫中确定了12个疫苗靶点 幼虫cDNA表达文库。该方法将是两个 传统的（重组产物的过表达）和 基于DNA，使用专有的Vical载体以及 几个重组载体，已显示出在其他方面的承诺， 系统.淋巴结转移诊断抗原的研究进展 还开展了防治丝虫病的工作。为此，cDNA表达 构建了来自雌性成虫的文库， 用适当的血清进行差异筛选。超过25 个别重组体已被鉴定为潜在的 诊断，表达和广泛的测试与大面板的 明确定义的血清已经鉴定出四种重组体， 对淋巴丝虫病患者具有特异性。序列 分析表明，3/4与 HMG样蛋白;一个是独特的，不同源的任何 各种数据库中的蛋白质。这种独特的多肽， Wb1.2和其中一种HMG样蛋白Wb5.4表达， 在几种不同的原核表达载体中高水平表达。 然后将部分纯化的蛋白质用作免疫印迹的基础 使用来自W. 班氏感染36例，B. B.马来感染（n=9）以及 来自非淋巴丝虫感染个体（n=27），以及 非丝虫性寄生虫感染（n=36）。正常人没有 还研究了寄生虫（n=15）。单独使用这些重组体 或组合使用时，灵敏度范围为65 - 98%， 特异性从89%到100%。此外， 已经合成了肽并用于绘制B细胞 这两种蛋白质的表位，希望利用 用于诊断未闭丝虫感染的基于肽的测定。