GLOBAL REGULATION OF VIRULENCE FACTOR IN BORDETELLA PERTUSSIS

百日咳杆菌的VIR基因座调控 多种毒力相关因子对环境的响应 刺激物。我们的研究最终是针对一种分子 环境信号产生的机制的表征 病毒调节基因表达的变化。公布的DNA 病毒基因座的序列预测有三种蛋白质，BvgA，BvgB，和 BvgC被编码，其中两个显示与一个家族的关系 细菌中称为“两个”的环境响应调节蛋白 组件系统。基因融合是通过使用 这使得我们能够在大肠杆菌中表达病毒编码的蛋白 以融合蛋白的形式与碱性磷酸酶结合。这些都是 用于研制定向的多克隆和单抗试剂 对抗BVG蛋白。这些抗体使我们能够检测到他们的 百日咳杆菌粗提物中的相应蛋白质。这些产品的使用 抗体工具使我们意识到BvgB和BvgC在 事实上是一种蛋白质，我们现在称之为BvgS(传感器)。原因 因为之前的误解是DNA序列中的一个错误 已经找到并确认了。我们现在有抗体试剂来对抗 VIR监管系统的两个组成部分，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 生化方面的。初步实验表明，该单抗 抗BvgS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 功能相当于多聚体，可能是二聚体。我们的抗体工具有 让我们开始通过使用生物化学来研究这个假说 化学交联剂在细菌膜制剂中的应用 百日咳。这些实验的初步证据支持我们的 假设BvgS作为二聚体是活跃的。

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.