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CLONING OF THE DUFFY BLOOD GROUP GENE

达菲血型基因的克隆

基本信息

批准号：
3355549
负责人：
A OSCAR POGO
金额：
$ 12.45万
依托单位：
NEW YORK BLOOD CENTER
依托单位国家：
美国
项目类别：
财政年份：
1987
资助国家：
美国
起止时间：
1987-07-01 至 1990-06-30
项目状态：
已结题

项目摘要

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.

Duffy血型基因的克隆是为了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寄生虫入侵。现在是克隆 Duffy系统由于抗原定义明确，基本上有四种主要的基因型，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处理而失活内切酶已经产生了单克隆抗体，其具有已经被用来开发一个程序，用于纯化达菲抗原首要任务是表征这种糖蛋白。这将是通过对与NH2相邻的氨基酸进行测序，末端以及内部多肽片段。选择多肽将用于化学合成寡脱氧核苷酸抗达菲多克隆抗体抗原将在兔子中产生，并将通过免疫化学方法通过亲和层析纯化。 Duffy mRNA的来源是人骨髓细胞、人网织红细胞和/或黑猩猩的骨髓细胞 Duffy mRNA将通过两个程序鉴定：免疫沉淀与兔多克隆抗体，在体外（网织红细胞裂解物）或体内合成的蛋白质（非洲爪蟾卵）翻译系统，和ii）在北方印迹中，用化学合成的探针探测mRNA样品寡脱氧核苷酸 cDNA文库将通过以下方法构建：使用Duffy mRNA的富集级分，合成双- 链cDNA并插入λ gt11和λ gt10中向量。 Duffy基因座的鉴定及等位基因的研究变体将通过从特定基因组库中筛选基因组库来完成，表型血型研究将通过经典的将研究血清学技术和提供信息的家庭先证者身份确认后，这些家族的达菲基因将被映射，并将在M13载体中亚克隆的DNA片段定序。本提案的长期目标是确定： Duffy蛋白中的糖苷化，ii）跨膜蛋白质的区域，和iii）蛋白质和糖在确定抗原变体和间日疟原虫疟疾受体