CLONING AND EXPRESSION OF THE HOOKWORM ANTICOAGULANT

钩虫抗凝剂的克隆和表达

• 批准号: 2517122
• 负责人: MICHAEL CAPPELLO
• 金额: $ 9.46万
• 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/2517122
• 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：计划项目的第一阶段包括休一个暑假 分子生物学课程和审计五门课程。第一阶段和第二阶段 将包括研究计划的不同部分。人体钩虫 感染的特征是慢性消化道出血和缺铁 贫血。成虫通过分泌下列物质促进血液摄取 一种名为钩虫外源性途径抑制物的有效抗凝血剂 (赫皮)。候选人已经分离出这种新的16 kDa蛋白质 从十二指肠钩虫的裂解物中提取。人工合成的寡核苷酸 从蛋白质的胰酶消化产生的氨基酸序列有 被用来产生和克隆含有部分 编码区序列。候选人计划克隆全长 从cdna文库中克隆基因，测序并表达重组 在细菌或哺乳动物载体系统中的HEPI。这一表达的蛋白质 将用于产生抗体以进行蛋白质的免疫定位 在生物体中，并用于蛋白质活性的生化分析。