PATHOGENESIS OF MEMBRANOUS GLOMERULONEPHROPATHY

描述(改编自《调查者摘要》)：调查者的 长期目标是了解发病机制。 膜性肾小球肾病(MGN)。他们将与被接受的人一起工作 MGN大鼠活动性Heymann肾炎(HN)模型及 他们将研究集中在假定的自身抗原gp330上，这是一个关键 参与发病机制。他们的具体目标是：a) 确定B细胞的位置和结构(氨基酸序列) Gp330的表位与该病有关。为了实现这一目标，他们将 分离与自身抗体反应的gp330蛋白水解性片段， 对片段进行微测序，将其定位于gp330序列，克隆到 通过聚合酶链式反应扩增得到相应的表达载体， 测试表达的融合蛋白以检测自身抗体的反应性 免疫印迹和ELISA法，以及免疫大鼠的免疫原性。他们 将缩小连续和不连续表位的位置(S) 通过分析PCR产生的较小克隆，合成重叠的多肽 跨越表位区域，并确定准确的氨基酸序列 自身抗体反应性线性表位(S)及其可能的序列 参与不连续的表位，并检测其致病潜能 所有已确定的表位。B)测试识别出的序列是否 活动性HN的耐受性和治疗潜力 推定的多肽(S)或蛋白质片段较大数量，给药 将这些多肽/蛋白质片段注入动物体内，然后评估疾病 参数。这些研究的数据将提供重要的新信息 活动性HN相关gp330 B细胞表位结构的研究 这一知识将有助于1)更好地理解发病机制 在分子水平上对这种疾病的治疗，2)可能用 特异性免疫调节。

DESCRIPTION (Adapted from Investigator's Abstract): The investigators' long-term goal is to understand the mechanisms involved in the pathogenesis of membranous glomerulonephropathy (MGN). They will work with the accepted experimental model of MGN, active Heymann Nephritis (HN) of rat, and concentrate their studies on the putative autoantigen, gp330, a key participant in the mechanisms of pathogenesis. Their specific aims are: A) Identify the location and structure (amino acid sequence) of the B-cell epitopes of gp330 involved in the disease. To achieve this they will isolate gp330 proteolytic fragments reactive with autoantibodies, microsequence the fragments, localize them in gp330 sequence, clone into expression vectors the corresponding cDNA's prepared by PCR amplification, test expressed fusion proteins for autoantibodies-reactivity by immunoblotting and ELISA, and for immunogenicity by immunizing rats. They will narrow down the location of the continuous and discontinuous epitope(s) by analyzing smaller clones produced by PCR, synthesize overlapping peptides spanning the epitope regions and identify the precise amino acid sequence of autoantibodies-reactive linear epitope(s) and the sequences possibly involved in the discontinuous epitopes, and test the pathogenic potential of all of the identified epitopes. B) Test the identified sequences for tolerogenic and therapeutic potential in active HN by synthesizing the putative peptide(s) or protein fragments in larger quantities, administering these peptide/protein fragments into animals, and then assessing the disease parameters. Data from these studies will provide important new information on the structure of the B cell epitopes of gp330 involved in active HN. This knowledge will lead to 1) a greater understanding of the pathogenesis of this disease at the molecular level and, 2) possibly its treatment with specific immunomodulation.