Identification of major outer surface proteins of Streptococcus agalactiae
Identification of major outer surface proteins of Streptococcus agalactiae
复制标题
DOI：
10.1128/iai.70.3.1254-1259.2002
复制
发表时间：
2002-03-01
影响因子：
3.1
通讯作者：
Santangelo, JD
中科院分区：
文献类型：
--
作者：
Hughes, MJG;Moore, JC;Santangelo, JD
为了鉴定无乳链球菌(B群链球菌)的主要外表面蛋白，进行了蛋白质组学分析。用双向电泳法分离了外表面蛋白的提取液。通过多肽测序和反向遗传方法相结合的方法识别可视化的斑点。在被确认为无乳链球菌特有的30个主要斑点中，已确定了27个。其中6个以前在无乳链霉菌中未鉴定的蛋白质被测序和克隆。它们是鸟氨酸氨基甲酰转移酶、磷酸甘油酸激酶、非磷酸化甘油醛-3-磷酸脱氢酶、嘌呤核苷磷酸化酶、烯醇化酶和葡萄糖-6-磷酸异构酶。使用革兰氏阳性表达系统，我们在体外系统中过度表达了其中两种蛋白质。这些重组、纯化的蛋白用于制备抗血清。通过抗血清对整个活细菌的反应能力，证实了这些蛋白质存在于外表面。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一种确定候选疫苗的技术的成功应用。
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.