CLONING OF THE DUFFY BLOOD GROUP GENE

点击翻译按钮获取中文摘要

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.