IDENTIFICATION OF GONOCOCCAL PH-REGULATED PROTEINS

淋病奈瑟菌产生保守和独特的热休克蛋白， 或应激蛋白，对热、营养缺乏和 厌氧微生物由于淋球菌暴露于pH的广泛变化， 它在体内感染的各种小生境，它可能会产生pH调节的压力 蛋白质也是。这项提案的目的是确定蛋白质 合成了N.淋病对pH应激的反应， 淋病患者的恢复期血清。实验方案 因为它们已经被成功地用来描述压力 其他致病菌的蛋白质。此外，这些协议是 非常简单，便于本科生参与。标准一- 二维电泳分析将用于检测 细胞质和外膜蛋白的诱导和抑制， 对体外pH变化的反应。然后，免疫印迹分离的外层 将探测在不同pH值下生长的生物体的膜蛋白 淋病患者的免疫血清 免疫印迹结果 实验将表明pH诱导的外膜蛋白是否 在淋球菌感染期间表达，以及是否为免疫显性。 最后，患者免疫系统识别的淋球菌pH调节蛋白 将血清纯化，印迹到聚偏二氟乙烯膜上， 并测序N-末端20-30个氨基酸。这些序列将是 与来自其他生物的应激蛋白氨基酸序列相比。 这些数据将使淋球菌pH调节蛋白的分类 作为高度保守的应激蛋白家族的成员，或作为 淋球菌独特的表面暴露应激蛋白会是极好的 淋球菌疫苗的候选人。这项研究将有助于 了解淋球菌应激蛋白在发病机制中的作用 和宿主的免疫反应

Neisseria gonorrhoeae produces conserved and unique heat shock proteins, or stress proteins, in response to heat, nutrient deprivation, and anaerobiasis. As the gonococcus is exposed to wide variations in pH in the various niches it infects in vivo, it may produce pH-regulated stress proteins as well. The objective of this proposal is to identify proteins synthesized by N. gonorrhoeae in response to pH stress that are recognized by convalescent serum from patients with gonorrhea. Experimental protocols were chosen as they have been used successfully to describe stress proteins in other pathogenic bacteria. Furthermore, these protocols are quite straightforward, enabling undergraduate participation. Standard one- and two-dimensional electrophoretic analyses will be used to detect induction and repression of cytoplasmic and outer membrane proteins in response to pH changes in vitro. Then, immunoblots of separated outer membrane proteins from organisms grown at different pHs will be probed with immune serum from patients with gonorrhea. Results of immunoblotting experiments will indicate whether pH-induced outer membrane proteins are expressed during gonococcal infection, and if they are immunodominant. Finally, gonococcal pH-regulated proteins recognized by patient immune serum will be purified, blotted onto polyvinylidene difluoride membranes, and the N-terminal 20-30 amino acids sequenced. These sequences will be compared to stress protein amino acid sequences from other organisms. These data will enable classification of gonococcal pH-regulated proteins as members of highly conserved stress protein families, or as unique to the gonococcus. Unique surface-exposed stress proteins would be excellent candidates for a gonococcal vaccine. This research will contribute toward an understanding of the role of gonococcal stress proteins in pathogenesis and the host immune response.