ENCYSTATION OF GIARDIA LAMBLIA

描述（改编自研究者摘要）：由于 细胞外囊壁是贾第虫病传播所必需的， 本项目的总体目标是深入了解 主要囊壁蛋白的结构和调控及其 加工和运输途径。 吉林医生已经完成了 循环G.首次在体外培养兰氏杆菌， 包囊形成需要表达两组受调节的抗原， 形成包囊特异性分泌囊泡（ESV）， 将它们转移到细胞表面。 单克隆抗体GCSA-1识别组 26、32和44千道尔顿（kD）的I蛋白，其在早期表达 在包囊中。 然而，只有32 kD的物质从孢囊中回收， 墙相比之下，第2组由四个WGA反应性囊肿壁组成， 糖蛋白（66-140 kD）在19小时后表达， 包囊形成。 针对纯化的gp 120的抗体识别蛋白质 两种主要调节蛋白的表位（120和85 kD）。 的 研究人员现在将根据这些重要发现， 囊壁形成的分子生化和细胞途径。 具体目标A是追踪ESV的起源、形成和交通 内容物和膜通过复杂的光和电子显微镜 技术，并询问两组囊壁抗原是否遵循 相同的运输和释放途径。 具体目标B是 比较这些包囊的表达、加工和释放的动力学 用脉冲追踪定量分析壁蛋白和糖蛋白 用抗GCSA-1或抗gp 120/85标记和免疫沉淀。 化学分析将揭示N-和O-糖基化的比例 并鉴定分离的GP 120和GP 85的糖。 在具体目标C中， 抗-GCSA-1和抗-gp 120/85将用于分离 编码GCSA-1基因和gp 120或gp 85的基因 图书馆 序列、拷贝数、包囊过程中的表达，以及 将分析这些基因的保守性。大调序列 囊壁蛋白和糖蛋白将提供重要的见解， 其结构和功能。 在特定目标D中，包囊细胞将 为了分离ESV并确定它们是否 含有她所描述的囊壁生物合成的酶。 此外，由于她已经发现二硫键在 她将确定这些结合物 form. 这些研究将大大增进我们对贾第虫的了解 包囊形成和囊壁形成，并可以指出 这一关键过程的中断，在未来。

DESCRIPTION (adapted from the investigator's abstract): Since the extracellular cyst wall is required for transmission of giardiasis, the overall goal of this project is to gain an in-depth understanding of the structure and regulation of the major cyst wall proteins and their processing and pathways of transport. Dr. Gillin has completed the life cycle of G. lamblia in vitro for the first time and shown that encystation entails expression of two groups of regulated antigens and formation of encystation- specific secretory vesicles (ESV) that deliver them to the cell surface. Monoclonal antibody GCSA-l recognizes Group I proteins of 26, 32, and 44 kilodalton (kD), which are expressed early in encystation. However, only the 32 kD species is recovered from cyst walls. In contrast, Group 2 consists of four WGA-reactive cyst wall glycoproteins (66-140 kD) that are expressed after 19 hours of encystation. Antibodies against purified gpl20 recognize, protein epitopes of both major regulated proteins (120 and 85 kD). The investigator will now build on these important findings to delineate key molecular biochemical and cellular pathways of cyst wall formation. Specific Aim A is to trace the origin, formation, and traffic of the ESV contents and membrane by sophisticated light and electron microscopic techniques and to ask whether the two groups of cyst wall antigens follow the same pathways of transport and release. Specific Aim B is to compare the kinetics of expression, processing and release of these cyst wall proteins and glycoproteins by quantitative analyses of pulse-chase labeling and immunoprecipitation with anti GCSA-l or anti-gp 120/85. Chemical analyses will reveal the proportion of N- and O-glycosylation and identify the sugars of isolated gpl20 and gp85. In Specific Aim C, anti-GCSA-l and anti-gp 120/85 will be used to isolate the genes that encode a GCSA-l gene and either gpl20 or gp85 from their expression library. The sequence, copy number, expression during encystation, and conservation of these genes will be analyzed. Sequences of the major cyst wall protein and glycoprotein will give important insights into their structures and functions. In Specific Aim D, encysting cells will be fractionated in order to isolate the ESV and determine whether they contain enzyme(s) of cyst wall biosynthesis, that she has described. Moreover, since she has found that disulfide bonds are important in the architecture of the cyst wall, she will determine where these bonds form. These studies will greatly enhance our understanding of giardia encystation and cyst wall formation and could point the way to interruption of this key process, in the future.