Molecular Definition Of Filarial And Related Nonfilarial Genes And Proteins

使用EST(表达序列标签)方法，我们已经能够在1687个EST的基础上从班氏五味子微丝虫中识别出超过965个基因簇。有趣的是，这些聚集的EST中有令人惊讶的高比例(584/965；60%)含有推测的信号肽，只有6个似乎是GPI锚定的。与马来丝虫(Brugia Malayi(BM))MFEST/簇相比，45%的WB簇与BM ESTs(同源性在86-100%之间)匹配，而更少(22%)与Ov(Ov)Mf ESTs(同源性在80%-100%之间)匹配。 利用马来丝虫噬菌体展示L3文库和已知人类受体的筛选，我们已经鉴定、克隆和鉴定了与人IL-5R、IL-10R和IL-13R结合的不同分子。我们称之为BM-IL5RBP的分子，马来丝虫IL5R结合蛋白以一种保持其与人类受体结合能力的方式高水平表达。针对预测免疫原肽的抗体抑制RBM-IL5BP的结合，并已被用来(通过免疫-EM)将BM-IL5RBP定位于感染期幼虫的表面。RBM-IL5BP本身不能延长人嗜酸性粒细胞的存活，但确实抑制了人IL5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的一种分泌产物，它对人嗜酸性粒细胞具有趋化作用。这种分泌产物通过G偶联受体CXCR3产生信号。 作为许多工作者联盟的一部分，我们已经参与了丝虫病基因组的注释，其草稿将于2007年9月发表。 一个单独的蛋白质组学分析也已经完成，使用质谱仪与整个阶段特定的胰酶消化相结合。不仅已经完成了成年雄性、成年雌性、微丝虫和感染性幼虫的阶段特异性蛋白质组，而且我们还完成了沃尔巴克氏布鲁氏菌蛋白质组和排泄/分泌蛋白质组。这些分析(仍在进行中)已经提供了明确的证据，证明那些编码“假设蛋白质”的基因是真实的，并且已经识别了所有预测的开放阅读框架中的近70%。 目前正在扩大对丝虫感染和类圆线虫的保护性免疫。用辐射幼虫免疫的小鼠或对SS感染表现出近100%保护作用的人的血清进行亲和纯化的HA抗原，鉴定了在大肠杆菌、酵母和杆状病毒中表达的6种候选抗原，以确定哪种产品可以大量生产，以便进行基于蛋白质的免疫实验。

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.