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Identification of Major Outer Surface Proteins of Streptococcus agalactiae

机译：无乳链球菌主要外表面蛋白的鉴定

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To identify the major outer surface proteins of Streptococcus agalactiae (group B streptococcus), a proteomic analysis was undertaken. An extract of the outer surface proteins was separated by two-dimensional electrophoresis. The visualized spots were identified through a combination of peptide sequencing and reverse genetic methodologies. Of the 30 major spots identified as S. agalactiae specific, 27 have been identified. Six of these proteins, previously unidentified in S. agalactiae, were sequenced and cloned. These were ornithine carbamoyltransferase, phosphoglycerate kinase, nonphosphorylating glyceraldehyde-3-phosphate dehydrogenase, purine nucleoside phosphorylase, enolase, and glucose-6-phosphate isomerase. Using a gram-positive expression system, we have overexpressed two of these proteins in an in vitro system. These recombinant, purified proteins were used to raise antisera. The identification of these proteins as residing on the outer surface was confirmed by the ability of the antisera to react against whole, live bacteria. Further, in a neonatal-animal model system, we demonstrate that some of these sera are protective against lethal doses of bacteria. These studies demonstrate the successful application of proteomics as a technique for identifying vaccine candidates.
机译：为了鉴定无乳链球菌（B组链球菌）的主要外表面蛋白，进行了蛋白质组学分析。外表面蛋白质的提取物通过二维电泳分离。通过结合肽测序和反向遗传学方法鉴定了可视化斑点。在确定为 S的30个主要景点中。目前已鉴定出27种无乳杆菌特异菌。这些蛋白质中有六个，以前在 S中无法识别。对无乳杆菌进行测序和克隆。这些是鸟氨酸氨基甲酰基转移酶，磷酸甘油酸激酶，非磷酸化3-磷酸甘油醛脱氢酶，嘌呤核苷磷酸化酶，烯醇酶和6-磷酸葡萄糖异构酶。使用革兰氏阳性表达系统，我们在体外系统中过表达了其中两种蛋白质。这些重组的纯化蛋白用于产生抗血清。通过抗血清对整个活细菌的反应能力，证实了这些蛋白质位于外表面。此外，在新生儿-动物模型系统中，我们证明了其中一些血清对致死剂量的细菌具有保护作用。这些研究证明了蛋白质组学作为鉴定候选疫苗的技术的成功应用。

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