STUDIES ON THE DUFFY BLOOD GROUP (FY) ANTIGEN

达菲血型（FY）抗原的研究

• 批准号: 6302332
• 负责人: A O POGO
• 金额: $ 23.65万
• 依托单位: NEW YORK BLOOD CENTER
• 依托单位国家: 美国
• 财政年份: 2000
• 资助国家: 美国
• 起止时间: 2000-02-15 至 2000-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6302332
• 关键词: blood group antigens; chemical binding; cytokine receptors; enzyme linked immunosorbent assay; erythrocyte membrane; gene expression; gene targeting; genetic mapping; genetically modified animals; human tissue; immunocytochemistry; immunoelectron microscopy; laboratory mouse; laboratory rabbit; malaria; messenger RNA; molecular shape; northern blottings; nucleic acid hybridization; nucleic acid sequence; organ; polymerase chain reaction; protein structure function; protozoal antigen; surface antigens

The Duffy blood group system consists of two major antigens Fya and Fyb produced by FY+A and FY+B co-dominant alleles. Antisera, anti-Fya and anti-Fyb define four phenotypes, Fy(a+b-), Fy(a-b+), Fy(a+b+), and Fy(a-b- ). Neither antiserum agglutinates Duffy Fy(a-b-) cells, the predominant phenotype in Blacks. Blacks with Fy(a-b-) erythrocytes cannot be infected by the human malarial parasite P. vivax and simian parasite P. knowlesi. We cloned the Duffy gene and showed that it encodes a glycoprotein (gp-Fy) of 337 residues. The objectives of this proposal are: (1) To determine the topology of the N- and C-terminal domains of gp-Fy, which will be done either by immunochemical binding of whole erythrocytes and inside-out membrane vesicles or double labeling immunoelectromicroscopy. (2) The determination, at the N-terminal domain, of the amino acids necessary for recognition sites of antibodies and malarial parasites. This will be carried out with ELISA assay of chemically synthesized peptides, followed by the construction of amino acid deletion and substitution mutants and expression in K562 cells. (3) The sequencing of non-erythroid gp-Fy mRNA in kidney, lung, thymus, spleen and brain. (4) The characterization by immunocytochemistry, of which cell(s) in these tissues produce gp-Fy. (5) Identification of the major ligand-binding requirements for chemokine recognition. This will be done by the construction of a panel of substitution and deletion mutants and expression in K562 cells. (6) The search for homologous FY in mouse will be done in a genomic library that will be screened with a probe containing either bone marrow cDNA or a probe having bone marrow and brain cDNA sequences. (7) To study interactions of Duffy and Rh protein since a Duffy antigenic determinant (Fy5) depends upon the presence of the Rh protein. The study will be done in Duffy-transfected K562 cells. The long-term goals of this project are to determine: (1) the gp-Fy protein function(s); (2) the significance of the Duffy-like protein in brain cells; (3) the sequence conformation that binds to the merozoites and the design of drugs which would block invasion; (4) whether gp-Fy protein is a transporter and finally, (5) the design of mouse knockout experiments to determine the significance of Duffy gene. This research project will have major biomedical consequences in the design of more sensitive and reliable reagents for the detection of Duffy antigens, anticipation of hemolytic diseases in the newborn, design of drugs to prevent parasite invasion, defining gp-Fy role as the human erythrocyte chemokine receptor and unraveling the function of Duffy gene. The Duffy gene is active in a variety of tissues, and these studies will establish its relevance in human biology.

达菲血型系统由两种主要抗原Fya和Fyb组成 由 FY+A 和 FY+B 共显性等位基因产生。 抗血清、抗 Fya 和 抗 Fyb 定义了四种表型：Fy(a+b-)、Fy(a-b+)、Fy(a+b+) 和 Fy(a-b-) ）。 抗血清均不凝集主要的 Duffy Fy(a-b-) 细胞 黑人的表型。 具有Fy(a-b-)红细胞的黑人不会被感染 由人类疟疾寄生虫间日疟原虫和猿猴寄生虫诺氏疟原虫引起。 我们克隆了 Duffy 基因并证明它编码糖蛋白 (gp-Fy) 337个残基。 本提案的目标是： (1) 确定 gp-Fy N 端和 C 端结构域的拓扑结构，这将完成 通过整个红细胞的免疫化学结合和由内而外 膜囊泡或双标记免疫电显微镜。 (2) 的 确定 N 端结构域所需的氨基酸 抗体和疟疾寄生虫的识别位点。 这将是 对化学合成肽进行 ELISA 测定，然后 通过构建氨基酸缺失和取代突变体以及 在K562细胞中表达。 (3)非红系gp-Fy mRNA测序 在肾、肺、胸腺、脾和脑中。 (4) 表征 免疫细胞化学，这些组织中的细胞产生 gp-Fy。 (5) 鉴定趋化因子的主要配体结合要求 认出。 这将通过建造一个面板来完成 取代和缺失突变体以及在 K562 细胞中的表达。 (6) 的 小鼠中同源 FY 的搜索将在基因组文库中进行 将用含有骨髓 cDNA 或 具有骨髓和脑cDNA序列的探针。 （七）学习 由于 Duffy 抗原决定簇，Duffy 和 Rh 蛋白之间的相互作用 (Fy5) 取决于 Rh 蛋白的存在。 研究将完成 在达菲转染的 K562 细胞中。 该项目的长期目标是 确定： (1) gp-Fy 蛋白功能； (二)意义 脑细胞中的类达菲蛋白； (3) 序列构象 与裂殖子结合以及阻断的药物设计 入侵； (4) gp-Fy 蛋白是否是转运蛋白，最后，(5) 设计小鼠基因敲除实验以确定其意义 达菲基因。 该研究项目将产生重大的生物医学影响 设计更灵敏、更可靠的试剂来检测 达菲抗原，新生儿溶血性疾病的预测，设计 预防寄生虫入侵的药物，将 gp-Fy 的作用定义为人类 红细胞趋化因子受体并揭示达菲基因的功能。 达菲基因在多种组织中都很活跃，这些研究将 确定其在人类生物学中的相关性。