Investigation of bacteriolytic enzyme effect to oral streptococci clinical isolation

口腔链球菌，尤其是变形链球菌和隐球菌，是引起龋齿的主要病原体。去年，我们发现了与s.a mutans有关的肽聚糖水解酶Aml，并体验了其对s.a mutans和s.a sobrinus的特异性裂解活性。该活性表明该物质对变形链球菌具有一定的抑制作用。今年，我们建立了大规模生产和精炼重组Aml蛋白的方法。其次，我们寻找影响因素，找到最佳的裂解活性条件。最后，研究了抗变形链球菌和sobrinus临床分离株的Aml裂解活性。1)重组蛋白AmlAt的批量生产和精制方法首先，我们鉴定了蛋白序列，得到了Aml的DNA信息。接下来，我们根据这些信息合成重组蛋白，并建立了批量生产方法和精炼方法。最后，我们从培养液中获得1mg的Aml重组蛋白。我们研究了影响Aml酶解活性的因素，发现Ca^<2+>和Na^+能增强Aml酶解活性。此外，triton-X也增强了裂解活性。最后，确定了最佳裂解条件，并配制了裂解缓冲液。（0.1M磷酸盐缓冲液，0.1M NaCl, 0.1mM CaCl_2, 0.1% triton-X）3)对变形链球菌和sobrinus临床分离株的Aml裂解活性研究我们分离了儿童临床分离株和sobrinus，得到了39株变形链球菌和9株sobrinus。接下来，我们研究了这些分离株在相同条件下的Aml裂解活性：添加10ug/ml Aml，孵育约90分钟。结果发现，所有分离株均具有较强的溶菌活性，但溶菌活性与龋病活性无相关性。

Oral Streptococci, especially S.mutans and S.sobrinus, are major pathogens causing dental caries. Last year, we identified Aml, the peptidoglycan hydrolase involved in S.mutans, and experienced its specific lytic activity against S.mutans and S.sobrinus. This activity showed some possibility of suppressing material against mutans streptococci.This year, we established mass-producing and refining method of recombinant protein Aml. Next, we searched for the influencing factors and found the best condition of lytic activity. And last, we investigated Aml lytic activity against S.mutans and S.sobrinus clinical isolates.1) Mass-producing and refining method of recombinant protein AmlAt first, we identified the protein sequence and got the DNA information about Aml. Next, we made up the recombinant protein from these informations, and we established mass-producing method and refining method. At last, we made 1mg Aml recombinant protein from the llitter cell culture.2) Characterization of AmlWe investigated the influencing factor of Aml lytic activity, and we found Ca^<2+> and Na^+ enhanced the activity. Moreover, triton-X also enhanced the lytic activity. Finally, we identified the best condition of lytic activity and made up the lysis buffer. (0.1M phosphate buffer, 0.1M NaCl, 0.1mM CaCl_2, 0.1% triton-X)3) Investigation of Aml lytic activity against S.mutans and S.sobrinus clinical isolatesWe separated the clinical isolation of S.mutans and S.sobrinus from children, and got the 39 S.mutans stocks and 9 S.sobrinus stocks. Next, we investigated the Aml lytic activity to these isolates in the same condition: added 10ug/ml Aml and incubated it for about 90 minutes. As a result, we found the strong lytic activity to all isolates, but there was no correlation between lytic activity and caries activity.