PILOT--CLONING/SEQUENCING OF CALMODULIN LIKE PROTEIN FROM BORDETELLA PERTUSSIS

中试——百日咳博德特氏菌钙调蛋白样蛋白的克隆/测序

• 批准号: 6325826
• 负责人: DELFINA C DOMINGUEZ
• 金额: $ 4.04万
• 依托单位: UNIVERSITY OF TEXAS EL PASO
• 依托单位国家: 美国
• 财政年份: 2000
• 资助国家: 美国
• 起止时间: 2000-06-01 至 2001-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6325826
• 关键词: Bordetella pertussis; adenylate cyclase; autoradiography; bacterial proteins; cAMP response element binding protein; calmodulin; electron microscopy; enzyme linked immunosorbent assay; genetic library; host organism interaction; molecular cloning; molecular pathology; nucleic acid sequence; polymerase chain reaction; posttranslational modifications; protein biosynthesis; protein sequence; spectrometry; virulence; western blottings

Bordetella pertussis, the causative agent of whooping cough, secrets multiple toxins which are presumed to be the cause of the systemic symptoms of the disease. Investigations on B. pertussis have shown that the bacterium produces an adenylate cyclase toxin which is activated by eukaryotic calmodulin. This adenylate cyclase toxin is one of the major virulence factors with B. pertussis. It is believed that the pathogenic mechanism involves the activation of the bacterial adenylate cyclase toxin by the host calmodulin causing over-production of cyclic-AMP in the host leading to alterations in cellular metabolism. Recently, biochemical experimentation has presented evidence of the existence of a calmodulin-like protein within B. pertussis and other bacteria. The purpose of this research is to elucidate the molecular details involved in the pathogenesis of B. pertussis infection. The proposed research will study the calmodulin-like protein present in B. pertussis by a) analyzing the chemical, physical and immunological properties of this protein, b) preparing oligonucleotide probes using the highly conserved region from residues 8-23 (which is identical in all calmodulins) to clone the gene, c) sequencing the cloned calmodulin gene and, d) evaluating the effects of this calmodulin on B. pertussis adenylate cyclase toxin. The long term goal of the research is to develop a more effective vaccine and to uncover novel developments towards the understanding of mechanisms of disease.

百日咳博德特氏菌是百日咳的病原体，它分泌多种毒素，这些毒素被认为是导致该疾病全身症状的原因。对百日咳博德特氏菌的研究表明，该细菌产生腺苷酸环化酶毒素，该毒素被真核钙调蛋白激活。这种腺苷酸环化酶毒素是百日咳博德特氏菌的主要毒力因子之一。据信，致病机制涉及宿主钙调蛋白激活细菌腺苷酸环化酶毒素，导致宿主体内环磷酸腺苷过量产生，从而导致细胞代谢的改变。最近，生化实验提供了百日咳博德特氏菌和其他细菌中存在类钙调蛋白的证据。本研究的目的是阐明百日咳博德特氏菌感染发病机制中涉及的分子细节。拟议的研究将通过以下方式研究百日咳博德特氏菌中存在的钙调蛋白样蛋白：a) 分析该蛋白的化学、物理和免疫学特性，b) 使用残基 8-23 的高度保守区域（与所有钙调蛋白）克隆该基因，c）对克隆的钙调蛋白基因进行测序，以及，d）评估该钙调蛋白对百日咳博德特氏菌腺苷酸环化酶毒素的影响。该研究的长期目标是开发一种更有效的疫苗，并发现在理解疾病机制方面的新进展。