MOLECULAR BIOLOGY, PLATELET AGGREGATION, S. SANGUIS

某些绿色链球菌(Agg+)菌株， 包括口腔共生植物群的主要成员， 血链球菌，在体外诱导人血小板聚集。什么时候 绿色链球菌进入人体后，它们可能会变得显著 某些严重人类疾病的病原体，甚至是病原体 与血小板相互作用，如细菌性心内膜炎、白塞氏病 症候群等 血链球菌的三种不同的表面抗原(adh+，Agg+)已经被 建议参与与血小板的相互作用，这是类 I类抗原(粘附素)，II类抗原，也称为 血小板聚集相关蛋白(PAAP)和III类抗原， 它具有胞外ATPase活性。血链球菌细胞通过与血小板结合 I类抗原，然后II类抗原触发并激活 致密颗粒的释放和血小板的聚集，类 III抗原通过水解从致密物质释放的ATP来放大反应 颗粒化成ADP。 尽管已经发现了各种无细胞形式的PAAP，但这些 分子不能直接诱导血小板聚集。直接证据 需要提供证明细胞表面的PAAP作为 激活和聚集血小板的独特因素。具体目标 到目前为止已经完成的这个项目的内容包括(1)开发 血链球菌PAAP等基因突变体的分离方法 转座子Tn916和等基因回复株，(2)克隆了PAAP基因(Paap) 从血链霉菌(S.sanguis)中分离纯化了部分重组蛋白。 克隆的PAAP(RPAAP)基因在大肠杆菌中的表达及比较 与从血链球菌分离的无细胞形式的PAAP在功能上， (4)对该基因进行了部分测序；(5)对该基因进行了特征分析 使用特定的测序软件分析测序数据 将该序列与基因库进行比较。还需要做进一步的研究 血链球菌基因特异性插入突变体的测序和制备。

Certain strains of viridans streptococci (Agg+), including the predominant member of the oral commensal flora, Streptococcus sanguis, induce human platelets to aggregate in vitro. When viridans streptococci entered the body, they may become significant pathogens or even etiological agents of certain severe human diseases by interacting with platelets, such as bacterial endocarditis, Behcet's syndrome, etc Three different surface antigens of S. sanguis (Adh+, Agg+) have been proposed to be involved in the interaction with platelets, which are class I antigen (adhesin), class II antigen, which is also called platelet-aggregation associated protein (PAAP), and class III antigen, which has ecto-ATPase activity. S. sanguis cells bind to platelets by class I antigen, and then class II antigen triggers and activates the release of the dense granules and the aggregation of platelets, the class III antigen amplifies the reaction by hydrolyzing ATP released from dense granules into ADP. Although various cell-free forms of PAAP have been identified, these molecules do not directly induce platelet aggregation. Direct evidence needs to be provided to prove that PAAP on the cell surface functions as an unique factor to activate and aggregate platelets. The specific aims of this project which so far have been accomplished includes (1) developed procedures for isolating an isogenic PAAP mutant of S. sanguis using transposon Tn916 and isogenic revertants, (2) cloned the PAAP gene (paap) from S. sanguis, (3) isolated and purified the portion of the recombinant PAAP (rPAAP) expressed by the cloned gene in E. coli and compared it functionally with the cell-free forms of PAAP isolated from S. sanguis, (4) partially sequenced the gene, and (5) characterized the gene by analyzing the sequencing data using specific sequencing software and comparing the sequence with genebanks. Further studies need to be done on sequencing and making a gene specific insertional mutant of S. sanguis.