CLONING OF THE DUFFY BLOOD GROUP GENE

建议克隆Duffy血型基因，以便 了解血型多样性的分子机制。 这种抗原的医学意义在于它参与了 间日疟原虫入侵。现在是克隆的好时机 Duffy系统因为抗原被很好地定义，所以有 基本上有四种主要的基因类型，该基因已被定位在 1号染色体和抗原决定簇可能是 蛋白质，因为它们通过蛋白水解物处理而失活 酵素。已经生产出一种单抗，它具有 已被用于开发达菲的纯化程序 抗原。 首要任务是确定这种糖蛋白的特性。这将是 通过对与NH2相连的氨基酸进行测序来完成 末端以及内部的多肽片段。已选择 多肽将用于化学合成 寡聚脱氧核苷酸。抗达菲多克隆抗体 抗原将在兔子体内产生，并将通过免疫化学方法 亲和层析纯化。Duffy mRNA的来源将 是人骨髓细胞、人网织红细胞和/或 黑猩猩的骨髓细胞。 Duffy mRNA将通过两个程序进行鉴定：i) 兔多克隆抗体免疫沉淀的初步研究 体外(网织红细胞裂解物)或体内合成的蛋白质 (非洲爪哇卵)的翻译系统，以及ii)在北缘的斑点 用化学合成的方法探测mRNA样本 寡聚脱氧核苷酸。将通过以下方式构建cDNA文库 利用Duffy信使核糖核酸的富集组分，合成双链 双链cDNA插入到gt11和gt10中 向量。Duffy基因座的鉴定及等位基因研究 变异将通过从特定基因中筛选基因组文库来完成 表型。血型研究将由经典 将研究血清学技术和提供信息的家系 在标的物确定之后。来自这些家庭的达菲基因 将被映射，并将DNA片段亚克隆到M13载体中 被测序。 这项提案的长期目标是确定：i)选址 Duffy蛋白中的糖苷作用，II)跨膜 蛋白质的区域，以及iii)蛋白质和糖在 间日疟原虫抗原变异株和间日疟原虫的检测 疟疾受体。

The cloning of the Duffy blood group gene is proposed in order to understand the molecular mechanism of blood group diversity. The medical significance of this antigen is its involvement in Plasmodium vivax parasite invasion. It is opportune to clone the Duffy system because the antigens are well defined, there are essentially four major genotypes, the gene has been mapped on chromosome 1 and the antigenic determinants are probably proteins since they are inactivated by treatment with proteolytic enzymes. A monoclonal antibody has been produced which has been used to develop a procedure for the purification of Duffy antigens. The first priority is to characterize this glycoprotein. This will be done by sequencing the amino acids contiguous to the NH2 terminus as well as internal polypeptide fragments. Selected polypeptides will be used to chemically synthesize oligodeoxynucleotides. Polyclonal antibodies against Duffy antigens will be produced in rabbits and will be immunochemically purified by affinity chromatography. Sources of Duffy mRNA will be human bone marrow cells, human reticulocyte and/or chimpanzee bone marrow cells. Duffy mRNA will be identified by two procedures: i) immunoprecipitation with the rabbit polyclonal antibodies in proteins synthesized in an in vitro (reticulocyte lysate) or in vivo (Xenopus eggs) translation system, and ii) in Northern blots of mRNA samples probed with chemically synthesized oligodeoxynucleotides. cDNA libraries will be constructed by using enriched fractions of Duffy mRNA, synthesizing double- stranded cDNA and inserting into lambda gtll and lambda gt10 vectors. Identification of the Duffy locus and studies of allelic variants will be done by screening genomic libraries from specific phenotypes. Blood group studies will be done by classical serological techniques and informative families will be studies after identification of propositi. Duffy genes from these families will be mapped and DNA fragments subcloned in M13 vectors will be sequenced. The long-term goals of this proposal are to determine: i) the sites of glycosidation in the Duffy protein, ii) the membrane spanning region of the protein, and iii) the role of the protein and sugars in determining the antigenic variants and the Plasmodium vivax malaria receptor.