CLONING AND EXPRESSION OF THE HOOKWORM ANTICOAGULANT

钩虫抗凝剂的克隆和表达

• 批准号: 2671368
• 负责人: MICHAEL CAPPELLO
• 金额: $ 9.52万
• 依托单位: YALE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1995
• 资助国家: 美国
• 起止时间: 1995-09-15 至 2000-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2671368
• 关键词: Baculoviridae; Escherichia coli; Nematoda; activation product; anticoagulants; blood coagulation; coagulation factor IX; coagulation factor VII; coagulation factor X; complementary DNA; genetic library; immunocytochemistry; in situ hybridization; monoclonal antibody; nucleic acid sequence; polymerase chain reaction; protease inhibitor; protein purification; protein sequence; protein structure function; recombinant proteins; thromboplastin; tissue /cell culture; transfection /expression vector; western blottings

DESCRIPTION: Phase I of the planned program consists of taking a summer course in molecular biology and auditing five courses. Phases I and II will include different portions of the research program. Human hookworm infection is characterized by chronic GI bleeding and iron deficiency anemia. The adult worm facilitates blood feeding through secretion of a potent anticoagulant called the Hookworm Extrinsic Pathway Inhibitor (HEPI). The candidate has already isolated this novel 16 kDa protein from lysates of Ancylostoma duodenale. Synthetic oligonucleotides from amino acid sequences generated from tryptic digests of the protein have been used to generate and clone a PCR product containing a partial coding region sequence. The candidate plans to clone the full length gene from a cDNA library, to sequence this and to express recombinant HEPI in a bacterial or mammalian vector system. This expressed protein will be used to generate antibody for immunolocallization of the protein in the organism and for biochemical assays of protein activity.

描述：第一阶段的计划包括采取一个夏天 分子生物学课程和审计五门课程。第一和第二阶段 将包括研究计划的不同部分。人钩虫 感染的特征是慢性胃肠道出血和缺铁 贫血成虫通过分泌 一种叫做钩虫外源性途径抑制剂的强效抗凝剂 （HEPI）。候选人已经分离出这种新的16 kDa蛋白质 来自钩虫的裂解物。合成寡核苷酸， 从蛋白质的胰蛋白酶解物产生的氨基酸序列具有 用于产生和克隆含有部分DNA片段的PCR产物。 编码区序列。候选人计划克隆全长 从cDNA文库中提取基因，对其进行测序并表达重组 在细菌或哺乳动物载体系统中的HEPI。表达的蛋白质 将用于产生用于蛋白质免疫定位的抗体 并用于蛋白质活性的生物化学测定。