GLOBAL REGULATION OF VIRULENCE FACTOR IN BORDETELLA PERTUSSIS

百日咳博德特氏菌vir基因座调节 多种与环境相关的因素 刺激。 我们的研究最终是针对 环境信号产生机制的表征 病毒调节基因表达的变化。 发表的DNA vir基因座的序列预测了三种蛋白质，BvgA，BvgB和 BvgC是编码的，其中两个显示与一个家族的相关性。 细菌中的环境响应调节蛋白被称为“两个 组件”系统。 基因融合是通过使用 PCR技术使我们能够在大肠杆菌中表达病毒编码的蛋白。杆菌 以碱性磷酸酶的融合蛋白的形式存在。 这些已经 用于开发多克隆和单克隆抗体试剂， 针对Bvg蛋白质。 这些抗体使我们能够检测它们的 粗B中的相应蛋白质。百日咳提取物。 使用这些 抗体工具使我们认识到，BvgB和BvgC是在 事实上，我们现在称之为BvgS（传感器）的蛋白质。 的原因 因为之前的误解是DNA序列中的错误， 已被定位并确认 我们现在有抗体试剂 这两个组成部分的vir监管系统，并正在使用这些 用于纯化这些蛋白质并测试其功能的试剂 生物化学 初步实验表明， 抗BvgS的抗体可用于免疫亲和纯化。 早期的遗传数据表明，传感器蛋白BvgS是 作为多聚体，可能是二聚体。 我们的抗体工具 让我们开始用生物化学的方法来研究这个假设， 化学交联剂对B的膜制剂。 百日咳。 这些实验的初步证据支持了我们的观点。 假设BvgS作为二聚体具有活性。

The vir locus of Bordetella pertussis regulates the expression of multiple virulence-associated factors in response to environmental stimuli. Our studies are ultimately aimed at a molecular characterization of the mechanism by which environmental signals result in changes in expression of vir-regulated genes. The published DNA sequence of the vir locus predicts that three proteins, BvgA, BvgB, and BvgC are encoded, two of which show relatedness to a family of environmentally responsive regulatory proteins in bacteria termed "two component" systems. Gene fusions have been created through the use of PCR which have enabled us to express the vir-encoded proteins in E. coli in the form of fusion proteins to alkaline phosphatase. These have been used to develop polyclonal and monoclonal antibody reagents directed against the Bvg proteins. These antibodies allow us to detect their corresponding proteins in crude B. pertussis extracts. The use of these antibody tools has led us to the realization that BvgB and BvgC are in fact one protein which we are now calling BvgS (for sensor). The reason for the previous misunderstanding was an error in the DNA sequence which has been located and confirmed. We now have antibody reagents against both components of the vir regulatory system and are using those reagents to purify these proteins and test their functions biochemically. Preliminary experiments show that the monoclonal antibody against BvgS will be useful in immunoaffinity purification. Earlier genetic data has suggested that the sensor protein BvgS is functional as a multimer, probably a dimer. Our antibody tools have allowed us to begin investigating this hypothesis biochemically by using chemical cross-linking reagents on membrane preparations of B. pertussis. Preliminary evidence from these experiments supports our hypothesis that BvgS is active as a dimer.