Development of methods for high-sensitivity-detection or removal of pathogens by utilizing defense systems of an invertebrate animal (horseshoe crab)

In order to develop new检测方法for β-glucan， with high sensitivity，这是一个独立的protease activity，we identified a β-glucan binding site in factor G. we expressed three kinds of domains in subunit α，in bacteria, as recombinant proteins. When β-glucan binding activity of the domains was examinedonly the xylanase Z-like domain, which is localized in the COOH-terminal end of the subunit，exhibited the activity.我们也可以examined inhibitory activity of the three domains on the activationof factor G by β-glucan. Againonly the xylanase Z-like domain showed inhibitory activity in a dose-dependent manner. Therefore，this domain to have binding activity to β-glucan，thus functioning as a competitor for factor G activity through its β-glucan-binding. We furthermoremeasured binding parameters of the domain and β-glucan by using a BIAcore equipment. the xylanaseZ-like domain showed a high Ka value of 8.03× D18 D1 M - d -1 - D1 against β-glucans.Since thexylanase Z-like domain is composed of two repeating units，each unit was separately expressed and measured their binding parameters. each repeating units alonetained the binding activity，but their binding constant (Ka) was much lower.In the present study，我们确定了一个小的protein fragment that the β-glucan binding activity and simultaneouslyestablished the方法to express the fragment as are recombinant protein in bacteria ina largequantity. We are currently developing new detection methods for fungus infection by detecting directinteraction for the fragment and β-glucan by a physicochemical方法。

In order to develop new detection methods for β-glucan, with high sensitivity, that is independent of the protease activity, we identified a β-glucan binding site in factor G. We expressed three kinds of domains in subunit α, in bacteria, as recombinant proteins. When β-glucan binding activity of the domains was examined, only the xylanase Z-like domain, which is localized in the COOH-terminal end of the subunit, exhibited the activity. We also examined inhibitory activity of the three domains on the activation of factor G by β-glucan. Again, only the xylanase Z-like domain showed inhibitory activity in a dose-dependent manner. Therefore, this domain was proved to have binding activity to β-glucan, thus functioning as a competitor for factor G activity through its β-glucan-binding. We furthermore measured binding parameters of the domain and β-glucan by using a BIAcore equipment. The xylanase Z-like domain showed a high Ka value of 8.03×10ィイD18ィエD1 MィイD1-1ィエD1 against β-glucans.Since the xylanase Z-like domain is composed of two repeating units, each unit was separately expressed and measured their binding parameters. Each repeating units alone retained the binding activity, but their binding constant (Ka) was much lower.In the present study, we identified a small protein fragment that has the β-glucan binding activity and simultaneously established the method to express the fragment as are recombinant protein in bacteria in a large quantity. We are currently developing new detection methods for fungus infection by detecting direct interaction for the fragment and β-glucan by a physicochemical method.