MOLECULAR GENETIC ANALYSIS OF MALARIA ANTIGENS

描述：疟疾是导致死亡和发病的主要原因之一。 全世界。为了继续制定新的更有效的控制策略，a 在分子水平上对寄生虫生命周期的详细了解是 危急时刻。从无性周期到性别分化的转变是 在实地传播疟疾所需的。一旦性发育是 开始后，寄生虫不再进行无性复制并死亡 成熟后几天，如果不是在血餐中被一只 蚊子。一旦被蚊子摄取，配子体就会被触发从 红细胞，如果受精，就会发育成感染性的子孢子。分子 参与这一复杂分化途径的机制主要是 未知，尽管它的形态特征和几个 性阶段特异性蛋白质已经被鉴定出来。特异性抗体 包括Pfs230和Pfs48/45在内的性期表面分子已被发现 为了阻止寄生虫感染蚊子的能力，从而阻止 疟疾传播。这些蛋白质多年来一直被研究为 候选疫苗，但它们的实际功能仍不清楚。 Pfs230和Pfs48/45仅在性分化过程中表达 人类宿主和通过寄生虫向蚊子的过渡 中肠。我们的假设是Pfs230和Pfs48/45在 配子体发育成可受精的受精卵 对它们表达的调控对它们的功能很重要。作为第一步 为了阐明Pfs230和Pfs48/45的功能，它们的表达 将被目标消失的干扰所抑制(特定目标1)及其效果 这对配子体和配子分化的影响将被评估(特定目标 2)。转化的寄生虫将根据抗药性进行选择、克隆和 对每个目标基因的干扰进行分析。形态和基因 将转化子和野生型寄生虫的表达模式进行比较 在整个性别分化过程中。以确认观察到的任何变化都是 对于靶基因的破坏，表达将通过以下方式恢复 互补性与表型再分析。Pfs230&的生物学作用 Pfs48/45还受其表达的时程和水平的影响。至 开始评估这一点，调节它们转录的元素将是 分析(具体目标3)。将对信使核糖核酸产生的时间进程进行评估 以及阶段性监管中涉及的5‘监管要素 将通过测试他们驱动特定阶段表达的能力来进行映射 氯霉素乙酰转移酶。确定的监管区域将是 用于测试核因子结合，以识别其他 基因，并构建包含特定阶段的、 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.