Technology for Analysis of Porphyromonas gingivalis

描述（由申请方提供）：牙龈卟啉单胞菌被认为是一种 是成人牙周病的重要病原体， 在某些系统性疾病如心血管疾病和低出生体重中。 本应用程序的目标是应用一种全新的方法， 鉴定牙龈卟啉单胞菌在体内而非体外表达的基因 增长这些基因可能对牙龈卟啉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），在许多方面上级其他相关技术，但 最重要的是，它不依赖于动物模型来模拟生长， 在人体内的病原体。IVIAT使用来自以下动物的合并血清中存在的抗体： 感染的患者作为探针来鉴定感兴趣的病原体基因。这 将在三个具体目标下实现。在第一个具体目标中， 来自牙周炎患者的血清将被体外完全吸附 生长的完整牙龈卟啉单胞菌细胞和细胞提取物。产生的血清将是 用于探测大肠杆菌中牙龈卟啉单胞菌的基因组表达文库。 coli-blotting方法。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， 阅读框（ORF）可能负责体内转录因子的表达， 诱导（IVI）抗原。相关的ORF将被亚克隆到 合适的表达载体和至少1 mg表达的蛋白质将 净化到同质。在Specific Aim 3中，给出了P. 牙龈炎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回收的牙龈卟啉单胞菌细胞进行， 用特异性针对IVI的标记抗体感染患者 抗原这些研究的结果有望提高对以下问题的认识： 牙龈卟啉单胞菌的致病机制， 常规方法无法发现的毒力相关基因。 这些基因和它们的产物是治疗和治疗癌症的极好候选者。 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.