Isolation of the Cytocidal toxin (CCT) from periodontopathic bacteria and antibody preparation against CCT: with a view to clinical application.

坦尼氏菌：一种重要的牙周病细菌，具有杀细胞毒素（TfCCT）、脂蛋白、唾液酸酶作为推定的毒力因子。其中，TfCCT可能在牙周炎的发生和发展中起重要作用，因此进一步研究该因子具有重要意义.杀细胞毒素（TfCCT）的纯化：通过DEAE-Sepharose柱层析和分子排阻层析，从TfCCT提取物中分离纯化TfCCT，在28kDa和400kDa以上的两个峰级分中检测到杀细胞活性。比较254 nm处的吸光度表明，该毒素可能与DNA相互作用。该毒素是一种DNA结合蛋白，通过肝素柱进一步纯化.抗杀细胞毒素单克隆抗体的制备：建立了能够用蛋白G-珠去除塔氏藻声波提取物中杀细胞毒性的抗杀细胞毒素单克隆抗体。作为结果， ...更多信息 t，鉴定出抗TfCCT抗体（Me-24）. CCT产生克隆的克隆：通过使用ZAP Express预消化的Gigapack克隆试剂盒构建T.lasthensis基因组文库。为了筛选，使用基于纯化的CCT蛋白的N-末端氨基酸序列的寡核苷酸变性DNA作为探针。测序结果表明，插入片段大小为2.1kbp，有4个开放阅读框（CCT 1、2、3、4）。CCT与prtH4基因序列同源性较高。对细胞周期的影响：在体外分离重组蛋白，并分析细胞周期停滞的影响或在每个细胞阶段的CyclinBi和p53水平。对染色的细胞进行流式细胞术。在G2期观察到高水平的p53和CyclinB1.临床应用用途：本菌被认为具有杀细胞毒素，在牙周炎患者中增加。本研究旨在探讨血清CCT抗体滴度及CCT蛋白水平与牙周炎临床症状的关系，以建立一种简便的牙周炎诊断方法（ELISA，Less

Tannetella forsythensis: one of the important periodontpathic bacteria which possesses the cytocidal toxin (TfCCT), lipoprotein, sialidase as a putative virulence factor. Among these, TfCCT might play an important role in initiation and progression of periodontitis and therefore it was of interest of further characterizing this factor.1. Purification of the cytocidal toxin (TfCCT): TfCCT was purified from T.forsythensis extract by using DEAE Sepharose column and size-exclusion chromatography, and cytocidal activity was detected in two peak fractions corresponding to 28 and over 400kDa. Comparing the absorbance at 254 nm suggest that this toxin may interact with DNA. This toxin is a DNA binding protein, further purification was carried out by heparmn column.2. Preparation of anti-cytocidal toxin monoclonal antibodies: Anti-cytocidal toxin monoclonal antibodies that are capable of removing cytocidal toxicity in Tforsythensis sonic extract with protein G-beads were established. As a resul … More t, anti-TfCCT antibody (Me-24) was identified.3. Cloning of CCT-producing clone: The T.forsythensis genoniic library was constructed by using ZAP Express predigested Gigapack cloning kits. For screening, the oligonucleotide degenerative DNA based on the N-terminal amino acid sequence of purified CCT protein was used as a probe. Sequencing of this clone revealed that the size of the insert fragment was 2.1 kbp and there were putative four ORFs (CCT1, 2, 3, and 4). There were high homology in sequence between CCT and prtH4. Evaluation of mechanisms of cytocidal activity: the effects to cell cycle: The recombinant proteins were isolated in vitro and were subjected to analyze the effect of cell cycle arrest or the levels of CyclinBi and p53 at each cell stage. Stained cells were subjected to flowcytometry. High levels of p53 and CyclinB1 were observed at G2 phase in cells treated with this toxin.5. Application for clinical use: This bacterium which has been supposed to have the cytocidal toxin increased in periodontitis patients. We are attempt to analyze the relationship between the levels of antibody titer against CCT or those of CCT protein and the clinical status of periodontitis for the purpose of establishing the simple examination of periodontitis (ELISA, Less