Genetical and biochemical study of neutrophil chemotactic inhibitor of bacteria as a juvenile periodontal pathogen

伴放线放线杆菌是青少年牙周炎的主要致病菌，可产生包括中性粒细胞趋化抑制因子在内的多种毒力因子，该因子于1991年报道，但至今尚未阐明。在本项目中，我们研究了该因子的优良性质，并克隆了该因子的编码基因。该因子是蛋白质类物质，耐热(90℃5分钟)，耐酸性、碱性和有机溶剂条件。我们以经热处理的胞浆组分为原料，采用TMS柱层析结合高效液相色谱分离纯化了该因子(约10K)。该因子N端20个氨基酸残基的序列分别与多杀性巴氏杆菌和流感嗜血杆菌的DNA结合蛋白HU相同和相似。由于伴生放线放线杆菌的DNA测序工作尚未完成，因此无法从DNA数据库中获得该细菌的HU编码DNA的全序列。然而，我们获得了该DNA的部分序列，并尝试利用PCR方法和菌落杂交技术进行了Hu基因的克隆，并尝试了重组Hu在大肠杆菌中的表达。我们进行了另一次抑制因子编码DNA的克隆，但没有成功。我们对含有该基因文库的重组大肠杆菌胞浆进行了活性筛选，发现了另一种独特的中性粒细胞趋化抑制因子。这是血根素H-11，是从中草药植物中分离得到的单宁。其抑制作用机制一直在研究中。

Actinobacillus actinomycetemcomitans is a major pathogen associated with juvenile periodontitis and produces a number of virulence factors including neutrophil chemotactic inhibitory factor which was reported in 1991 but has not so far been clarified. In this project we to study the fine properties of the factor and clone the gene encoding the factor. This factor is proteinous, heat-stable (90 ℃ 5 min), and resistant for acidic, basic, and organic solvent-containing conditions. We purified the factor (Mw ; about 10K) using TMS-column chromatography with HPLC from the heat-treated cytosolic fraction as a starting material. The sequence of N-terminal 20 amino acid residues of the factor is identical and similar to those of DNA-binding protein HU of Pasteurella multocida and Haemophilus influenzae, respectively. We could not obtain the complete sequence of DNA encoding HU of A. actinomycetemcomitans from DNA database because the DNA sequencing project for this bacterium dose not finish. However, we obtained partial sequence of the DNA, and we try to carry out the cloning the HU gene using PCR method and colony hybridization technique, and also we try the expression of recombinant HU in Escherichia coli.We performed another cloning procedure for inhibitory factor-encoding DNA, but did not succeed. We screened the activities of cytosol of recombinant E. coli containing the gene library on the vector plasmids.We found the another unique inhibitor for the neutrophil chemotaxis. This is sanguiin H-11 , a tannin isolated from Chinese medicinal plant. The mechanism of the inhibition of has been studying.