MOLECULAR GENETIC ANALYSIS OF MALARIA ANTIGENS

描述：疟疾是死亡和发病的主要原因之一 国际吧为了继续制定新的、更有效的控制战略， 在分子水平上详细了解寄生虫的生命周期， 很危险从无性生殖周期到性分化的转变是 疟疾在野外传播所需的量。一旦性发育 一旦启动，寄生虫不再进行无性复制并死亡， 在达到成熟后的几天内，如果没有被吸血鬼吸收， 蚊子一旦被蚊子摄入，配子体就会被触发， 如果受精，则发育成感染性子孢子。分子 参与这一复杂分化途径的机制主要是 未知，虽然它已被表征形态和几个 性阶段特异性蛋白已被鉴定。特异性抗体 有性阶段的表面分子，包括Pfs 230和Pfs 48/45，已经被证明是 阻断寄生虫感染蚊子的能力，从而阻断， 疟疾传播。这些蛋白质已经研究了很多年， 疫苗候选人，但其实际功能仍然未知。 Pfs 230和Pfs 48/45都只表达在性分化过程中， 通过寄生虫进入蚊子体内 中肠我们的假设是，Pfs 230和Pfs 48/45在这一过程中起重要作用。 配子母细胞发育成能存活的受精卵， 调节它们的表达对于它们的功能是重要的。作为第一步 为了阐明Pfs 230和Pfs 48/45的功能， 将受到目标破坏（具体目标1）的抑制， 这对配子母细胞和配子分化的影响将被评估（具体目标 2）。转化的寄生虫将通过抗药性选择，克隆， 分析每个靶基因的破坏。形态和基因 将比较转化体和野生型寄生虫的表达模式 在性别分化中。确认观察到的任何变更 到靶基因的破坏，表达将被恢复， 互补并重新分析表型。Pfs 230的生物学作用& Pfs 48/45也受其表达的时间过程和水平的影响。到 开始评估这一点，调控其转录的元件将是 具体目标（3）。将评估mRNA产生的时间过程 以及参与阶段特异性调控的5'调控元件 将通过测试它们驱动特定阶段表达的能力来映射， 氯霉素乙酰转移酶确定的监管区域将包括 用于检测核因子结合，以识别其他细胞中的类似区域， 基因，并构建含有阶段特异性， 诱导型启动子

DESCRIPTION: Malaria is one of the major causes of mortality and morbidity worldwide. To continue to formulate new, more effective control strategies, a detailed understanding of the parasite life cycle on the molecular level is critical. The transition from the asexual cycle to sexual differentiation is required for malaria transmission in the field. Once sexual development is initiated, the parasite no longer undergoes asexual replication and dies several days after reaching maturity if not taken up in a blood meal by a mosquito. Once ingested by a mosquito, gametocytes are triggered to emerge from the RBC and, if fertilized, develop into infectious sporozoites. The molecular mechanisms involved in this complex differentiation pathway are largely unknown, although it has been characterized morphologically and several sexual-stage specific proteins have been identified. Antibodies specific for sexual-stage surface molecules, including Pfs230 and Pfs48/45, have been shown to block the ability of the parasite to infect mosquitoes, thus blocking, malaria transmission. These proteins have been studied for many years as vaccine candidates but their actual functions remain unknown. Both Pfs230 and Pfs48/45 are expressed only during sexual differentiation in the human host and through the transition of the parasite into the mosquito midgut. Our hypothesis is that Pfs230 & Pfs48/45 play a significant role in the development of gametocytes into viable fertilized zygotes and that the regulation of their expression is important to their function. As a first step toward the elucidation of the function of Pfs230 & Pfs48/45, their expression will be inhibited by targeted-gone disruption (Specific aim 1) and the effects this has on gametocyte & gamete differentiation will be evaluated (Specific aim 2). Transformed parasites will be selected by drug- resistance, cloned, and analyzed for disruption of each targeted gene. The morphology and gene expression pattern of transformants and wild-type parasites will be compared throughout sexual differentiation. To confirm that any changes observed are due to disruption of the targeted-gene, expression will be restored by complementation and the phenotype reanalyzed. The biological role of Pfs230 & Pfs48/45 is also affected by the time course and level of their expression. To begin to evaluate this, the elements regulating their transcription will be analyzed (Specific aim 3). The time course of MRNA production will be evaluated and the 5' regulatory elements that are involved in stage- specific regulation will be mapped by testing their ability to drive stage-specific expression of chloramphenicol acetyltransferase. The regulatory regions identified will be used to test for nuclear factor binding, to identify similar regions in other genes, and to construct transformation plasmids containing stage-specific, inducible promoters.