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

利用无脊椎动物（鲎）的防御系统开发高灵敏度检测或去除病原体的方法

基本信息

批准号：
09558087
负责人：
MUTA Tatsushi
金额：
$ 6.46万
依托单位：
KYUSHU UNIVERSITY
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (B)
财政年份：
1997
资助国家：
日本
起止时间：
1997 至 1999
项目状态：
已结题

项目摘要

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.

为了开发新的检测β-葡聚糖的方法，具有高灵敏度，即不依赖于蛋白酶活性，我们确定了G因子中的β-葡聚糖结合位点。我们在细菌中表达了α亚基中的三种结构域作为重组蛋白。当检查结构域的β-葡聚糖结合活性时，仅位于亚基的COOH末端的木聚糖酶Z样结构域表现出活性。我们还检测了这三个结构域对β-葡聚糖激活G因子的抑制活性。同样，仅木聚糖酶Z样结构域以剂量依赖性方式显示抑制活性。因此，该结构域被证明具有与β-葡聚糖的结合活性，从而通过其β-葡聚糖结合作为G因子活性的竞争者。我们还通过使用BIAcore设备测量了结构域和β-葡聚糖的结合参数。木聚糖酶Z样结构域对β-葡聚糖的Ka值为8.03×10 - 18 kDa D1 M kDa D1-1 kDa D1，由于木聚糖酶Z样结构域由两个重复单元组成，因此分别表达每个单元并测定其结合参数。在本研究中，我们鉴定了一个具有β-葡聚糖结合活性的小片段蛋白，同时建立了在细菌中大量表达该片段的重组蛋白的方法。我们目前正在开发新的检测真菌感染的方法，通过检测片段和β-葡聚糖的直接相互作用的物理化学方法。