Proteomic Analysis of Hookworm Larval Activation

描述(申请人提供)：钩虫感染仍然是世界上最重要的传染病之一，全世界有超过8亿人感染。目前的控制策略成效有限，需要新的疫苗抗原和药物靶点。在感染最终宿主期间，钩虫的第三阶段感染幼虫(L3)遇到宿主介导的信号，重新激活其受阻的发育程序。这些发育计划的执行最终导致在小肠中建立成虫。从自由生活的L3到寄生的L3的这种转变在寄生学中是一个至关重要的事件。尽管它起着核心作用，但人们对这种“向寄生的转变”的分子生物学知之甚少。以恢复摄食为激活标志的体外实验表明，激活需要信使核糖核酸和蛋白质合成。对未激活的、激活的和环己酰亚胺抑制的L3进行的双向凝胶电泳显示，大约有25种蛋白质在激活的前2小时经历了表达的变化。在这项研究中，将使用2D-Gel电泳法和LC/MS/MS来鉴定这些发育调节蛋白。从凝胶中提取的蛋白质用胰酶消化，用LC/MS/MS进行测序，蛋白质序列数据用于搜索钩虫表达序列标签数据库，并通过聚合酶链式反应或文库筛选克隆相应的cDNA。在没有钩虫EST序列的情况下，根据反向翻译的蛋白序列设计简并引物，通过低严格的PCR扩增cDNAs。一旦分离出cDNA序列，将通过定量逆转录聚合酶链式反应(Q-RT-PCR)确定蛋白质的表达模式。将建立每个蛋白质的TaqMan分析，并通过实时定量PCR确定每个基因在激活过程中的表达模式。与向寄生的转变相关的蛋白质的鉴定和特征将提供有关寄生所涉及的分子过程的第一批信息，包括对感染至关重要的蛋白质。这些蛋白是预防钩虫感染和控制钩虫疾病的潜在疫苗抗原和药物靶点。

DESCRIPTION (provided by the applicant): Hookworm infection continues to rank among the world's most important infectious diseases, with over 800 million people infected worldwide. Current control strategies are of limited success, and new vaccine antigens and drug targets are needed. During infection of the definitive host, the third-stage infective larva (L3) of hookworms encounter a host-mediated signal that re-activates its arrested developmental programs. Execution of these developmental programs culminates in the establishment of adult worms in the small intestine. This transition from the free-living L3 to the parasitic L3 is a critically important event in parasitism. Despite its central role, very little is known about the molecular biology of this "transition to parasitism". An in vitro assay employing the resumption of feeding as a marker for activation was used to demonstrate that mRNA and protein synthesis are required for activation. Two-dimensional-gel electrophoresis of non-activated, activated, and cycloheximide inhibited L3 revealed approximately 25 proteins that undergo expression changes during the first 2 hrs of activation. In this study, 2D-gel electrophoresis and liquid chromatography tandem mass spectroscopy (LC/MS/MS) will be used to identify these developmentally regulated proteins. Proteins excised from the gels will be digested with trypsin and sequenced by LC/MS/MS. Protein sequence data will be used to search hookworm expressed sequence tagged databases, and the corresponding cDNAs cloned by PCR or library screening. In the absence of a hookworm EST sequence, degenerate primers designed from the back-translated protein sequence will be used to amplify the cDNA by low stringency PCR. Once the cDNA sequences have been isolated, the expression pattern of the proteins will be determined by quantitative reverse transcription PCR (Q-RT-PCR). TaqMan assays for each protein will be developed and used to determine the expression pattern of each gene during the activation process by real-time quantitative PCR. The identification and characterization of the proteins associated with the transition to parasitism will provide the first information about the molecular processes involved in parasitism, including proteins critical for infection. These proteins represent potential vaccine antigens and drug targets for the prevention of hookworm infection and the control of hookworm disease.