Molecular ID Of Filarial/Nonfilarial Genes/Proteins

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 BP [马来丝虫IL 5结合蛋白]的分子以保持其与人受体结合的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，并已用于将Bm-IL 5 BP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 使用第一代马来丝虫表达微阵列，我们已经能够（不令人惊讶地）鉴定大量成年男性和成年女性特异性转录物，并使用定量RT-PCR证实其表达（Ndi et al，未发表）。在使用有价值的L3和其他丝虫RNA进行特定实验之前，我们等待第二代微阵列（含有>12000个寡核苷酸）。 对粪类圆线虫采取了类似的方法，从两个独立的生命周期阶段进行了>10000个EST。将该寄生线虫与自由生活的C. elegans已经完成;数据表明寄生和非寄生线虫之间基因表达的巨大差异（Mitreva，DM，McCarter，JP. Marin，J，Dante，M，Wylie，T，Chipelli，B，Pape，D，克利夫顿，SW. Nutman，TB，Waterston，RH.寄生线虫和自由生活线虫Strongyloides stercoralis和秀丽隐杆线虫基因表达的比较基因组学。Genome Res 14：209-220，2004）。 一个单独的蛋白质组学方法的保护性免疫正在进行平行的基因组方法。抗原亲和纯化使用血清从任一小鼠免疫与辐射幼虫，显示接近100%的保护挑战感染与S。类圆线虫或对Ss感染表现出免疫力的人使用与质谱和/或内部蛋白质序列分析偶联的2D-PAGE进行分析。在鉴定的这些蛋白质中，已经表征和克隆了四种（Ss-eat-6、Ss-tpn-1、Ss-xxx、Ss-xxx）。第一，Ss-eat-6已被证明在接种含有Ss-eat-6的DNA疫苗的小鼠中诱导适度的保护

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-IL5BP [Brugia malayi IL5 binding 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-IL5BP 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 Using the first generation Brugia malayi expression microarrays, we have been able (not surprisingly) to identify a large number of adult male- and adult female-specific transcripts and corroborate their expression using quantative RT-PCR (Ndi et al, unpublished). We await the second generation microarray (containing >12000 oligos) before proceeding with specific experiments using valuable L3 and other filarial RNA. A similar approach has been taken with Strongyloides stercoralis with >10000 ESTs being done from two separate lifecylcle stages. Comparisons between this parasitic nematode and the free-living C. elegans has been perfomed; the data demonstrate the vast differences in gene expresssion between parasitic and non-parasitic nematodes (Mitreva, DM, McCarter, JP. Marin, J, Dante, M, Wylie, T, Chipelli, B, Pape, D, Clifton, SW. Nutman, TB, Waterston, RH. Comparative Genomics of Gene Expression in the Parasitic and Free-living Nematodes Strongyloides stercoralis and Caenorhabditis elegans. Genome Res 14: 209-220, 2004). A separate proteomic approach to protective immunity is being performed in parallel to the genomic approach. 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 are being analyzed using 2D-PAGE coupled to mass spectroscopy and/or internal protein sequence analyses. Among those proteins identified, four have been characterized and cloned (Ss-eat-6, Ss-tpn-1, Ss-xxx, Ss-xxx). One, Ss-eat-6 has been shown to induce modest protection in mice vaccinated with a DNA vaccine containing Ss-eat-6