Proteomic Analysis of Hookworm Larval Activation

描述（由申请方提供）：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。目前的控制策略是有限的成功，需要新的疫苗抗原和药物靶点。在最终宿主的感染过程中，钩虫的第三阶段感染性幼虫（L3）遇到宿主介导的信号，重新激活其停滞的发育程序。这些发育程序的执行最终在小肠中建立蠕虫。这种从自由生活的L3到寄生的L3的转变是寄生中至关重要的事件。尽管它的核心作用，很少有人知道这种“过渡到寄生”的分子生物学。采用恢复进食作为激活标志物的体外测定用于证明激活需要mRNA和蛋白质合成。二维凝胶电泳的非活化，活化，放线菌酮抑制L3显示约25蛋白，在第一个2小时的活化过程中经历的表达变化。在这项研究中，二维凝胶电泳和液相色谱串联质谱（LC/MS/MS）将被用来确定这些发育调控蛋白。将从凝胶中切下的蛋白质用胰蛋白酶消化，并通过LC/MS/MS进行测序。蛋白质序列数据将用于搜索钩虫表达的序列标记数据库，并通过PCR或文库筛选克隆相应的cDNA。在没有钩虫EST序列的情况下，将使用从反向翻译的蛋白质序列设计的简并引物通过低严格PCR扩增cDNA。一旦分离出cDNA序列，将通过定量逆转录PCR（Q-RT-PCR）确定蛋白质的表达模式。将开发每种蛋白质的TaqMan检测试剂盒，并通过实时定量PCR确定激活过程中每种基因的表达模式。鉴定和表征与过渡到寄生相关的蛋白质将提供有关寄生的分子过程的第一个信息，包括对感染至关重要的蛋白质。这些蛋白质代表了预防钩虫感染和控制钩虫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.