LOW PASSAGE ASSOCIATED PROTEINS FORM B BURGDORFERI

伯氏芽孢杆菌外表面蛋白对细菌生长发育至关重要 确定感染的细菌将是很好的候选疫苗 用于预防莱姆病。我们实验室已经开发出两种方法 对于识别和定位这些潜在的重要 蛋白质，我们开发了：(1)一种免疫吸收技术；(2)一种 平衡蔗糖密度膜分离技术 离心法。用第一种方法，我们鉴定了十种蛋白质，它们是 仅在低传代、强毒力伯氏杆菌B3I株中表达。与 第二种方法，我们纯化了伯氏杆菌的外膜 结合这些技术，我们已经识别出两个(58和33 kDa)低- 与通道相关的外膜蛋白。两者似乎都是以前的 未鉴定的蛋白质和初步数据表明，其中一种是58 kDa 蛋白质，可能在伯格多芬转铁蛋白的利用中起作用。在……里面 为了进一步鉴定这些蛋白质：(1)我们将鉴定 含有编码58 kDa和33 kDa低通道基因的重组克隆 我们之前已经确定的相关外膜蛋白。我们 将使用蛋白质表达系统和 序列分析。(2)我们将开始对58 和33 kDa的蛋白质。我们将构建基因融合以纯化 重组58 kDa和33 kDa蛋白。纯化的蛋白质将用于 在小鼠身上进行的保护研究。(3)分离伯氏假单胞菌突变体 不表达低通道相关的58和33 kDa蛋白和 评估它们在致病力中的作用。最近描述的两种方法将是 使用：(A)逃逸变异技术；和(B)基因破坏技术。

Outer surface proteins of Borrella burgdorferi that are essential for the bacterium to establish an infection would be excellent vaccine candidate for prevention of Lyme disease. Our laboratory has developed two methods for the identification and localization of these potential important proteins, We have developed: (1) an immune absorption technique; and (2) a membrane separation technique using equilibrium sucrose density centrifugation. With the first method, we identified ten proteins that are expressed only in low-passage, virulent B. burgdorferi strain B3I. With the second method, we have purified outer membranes of B. burgdorferL By combining these techniques, we have identified two (58- and 33-kDa) low- passage-associated outer membrane proteins. Both appear to be previously uncharacterized proteins and preliminary data suggests that one, the 58-kDa protein, may play a role in transferrin utilization by B. burgdorfen. In order to further characterize these proteins: (1) We will identify recombinant clones harboring genes encoding the 58-and 33-kDa low-passage- associated outer membrane proteins that we have previously identified. We will characterize positive clones using protein expression systems and sequence analysis. (2) We will begin the initial characterization of the 58 and 33-kDa proteins. We will construct gene fusions in order to purify recombinant 58- and 33-kDa proteins. Purified protein will be used for protection studies in mice. (3) We will isolate mutants of B. burgdorfeti that do not express low-passage-associated 58- and 33-kDa proteins and assess their role in virulence. Two recently described methods will be used: (a) the escape variant technique; and (b) gene disruption technique.