Technology for Analysis of Porphyromonas gingivalis

描述(申请人提供)：牙龈卟啉单胞菌被认为是 成人牙周病的重要病原体，也牵涉其中 在一些系统性疾病中，如心血管疾病和低出生体重。 此应用程序的目标是将一种全新的方法应用于 确定牙龈假单胞菌在体内表达但不在体外表达的基因 成长。这些基因可能对牙龈假单胞菌的能力很重要。 导致疾病。这种方法被称为体内诱导抗原技术 (IVIAT)，在许多方面优于其他相关技术，但 最重要的是，它不依赖动物模型来模拟生长 人类身上的病原体。IVIAT使用存在于混合血清中的抗体 以感染患者为探针，确定感兴趣的致病基因。这 将通过三个具体目标来实现。在第一个具体目标中，汇集了 牙周炎患者的血清在体外将被完全吸附 培养完整的牙龈假单胞菌细胞和细胞提取物。由此产生的血清将是 用于探索牙龈假单胞菌在大肠杆菌中的基因组表达文库。 用菌落印迹法检测了该菌的表达。在特定目标2中，克隆的DNA插入 将对反应性克隆进行测序和分析，以确定克隆的开放程度 可能负责体内表达的阅读框架(ORF) 诱导(IVI)抗原。相关ORF将被亚克隆到一个 合适的表达载体和至少1毫克的表达蛋白 被提纯到同质。在具体目标3中，给出了P. 牙龈杆菌在实际感染过程中会产生IVI抗原 通过探测从牙菌斑样本中提取的牙龈假单胞菌细胞进行 感染了针对IVI的标记抗体的患者 抗原。这些研究的结果有望提高对 牙周炎假单胞菌致病机制的研究 传统方法无法发现的毒力相关基因。 这些基因及其产物是治疗和治疗的极佳候选者 诊断目标和疫苗设计。

DESCRIPTION (provided by applicant): Porphyromonas gingivalis is considered an important etiologic agent of adult periodontal diseases and is also implicated in some systemic diseases such as cardiovascular disease and low birth weight. The goal of this application is to apply a fundamentally new approach to identify P. gingivalis genes that are expressed during in vivo but not in vitro growth. Such genes are likely to be important to the ability of P. gingivalis to cause disease. The approach, called In Vivo Induced Antigen Technology (IVIAT), is superior to other related technologies in a number of ways, but most importantly because it does not rely on animal models to mimic the growth of the pathogen in humans. IVIAT uses antibodies present in pooled sera from infected patients as probes to identify the pathogen genes of interest. This will be accomplished in three Specific Aims. In the first Specific Aim, pooled sera from periodontitis patients will be exhaustively adsorbed with in vitro grown whole P. gingivalis cells and cell extracts. The resulting serum will be used to probe a genomic expression library of P. gingivalis in Escherichia. coli using colony-blotting methods. In Specific Aim 2, the cloned DNA inserts in reactive clones will be sequenced and analyzed to determine the cloned open reading frames (ORFs) likely to be responsible for expression of the in vivo induced (IVI) antigens. The pertinent ORFs will be subcloned into an appropriate expression vector and at least 1 mg of the expressed protein will be purified to homogeneity. In Specific Aim 3, a direct proof that P. gingivalis produces IVI antigens during an actual infectious process will be performed by probing P. gingivalis cells recovered from plaque samples of infected patients with labeled antibodies specifically directed against the IVI antigens. The results of these studies are expected to improve understanding of the pathogenic mechanisms employed by P. gingivalis by identifying virulence-associated genes that would not be found by conventional methods. These genes and their products are excellent candidates for therapeutic and diagnostic targets and for vaccine design.