Development of a Reagent to Detect and Quantify Minute Amount of Cell Wall Components of Bacteria and Fungi.

开发一种试剂来检测和定量细菌和真菌的微量细胞壁成分。

• 批准号: 63840022
• 负责人: ASHIDA Masaaki
• 金额: $ 8.26万
• 依托单位: The Institute of Low Temperature Science, Hokkaido University
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Developmental Scientific Research (B).
• 财政年份: 1988
• 资助国家: 日本
• 起止时间: 1988 至 1990
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-63840022/
• 关键词: Peptiaoglycan; Beta-1, 3-glucan; Lypopolysaccharide; Limulus test; Insect; Hemolymph; βー1,3ーグルカン; カスケ-ド; 汚染検査; 昆虫血液; バクテリア; カビ; フェノ-ル酸化酵素; β-1,3-グルカン; 異物認識; フェノール酸化酵素

The Limulus test is widely used to detect pollution of medical instruments with bacteria and fungi. However, the test method has drawbacks such as following : Peptidoglycan can not be detected by Limulus test ; As lipopolysaccharide and beta-1, 3-glucan are positive in the test, it cannot be differentiated whether the detected pollution is due to that of bacteria or fungi ; Hcmocyte lysate of horseshoe crab is employed to the Limulus test, leaving the possibility that we may not have enough supply with horseshoe crab in future.The purpose of this research project is to develop a commercially available reagent to specifically detect microbial cell wall components. The silkworm (Bombyx mori) plasma contains prophenoloxidase cascade which is triggered by peptidoglycan and beta-1, 3-glucan. We intended to use the cascade to detect pollution of medical instruments with bacteria and fungi. We succeeded in developing a method to quantify peptidoglycan or beta-1, 3-glucan at very low concentrations like several pg/ml. Furthermore, we could make silkworm prophenoloxidase cascade specific to peptidogly can by using antibody against beta-1, 3-glucan recognition protein, which is a component of prophenoloxidase cascade and specifically interects with beta-1, 3-glucan. These results on the studies of prophenoloxidase cascade indicated that the cascade is a good candidate as a reagent for detection and quantification of microbial pollution of medical instruments, especially for detection of Gram-positive bacteria. We are in the process to develop methods to collect large quantity of silkworm plasma of which cascade is not triggered and to stabilize the cascade in the plasma in order that the plasransported from supplier to users.

鲎试验广泛用于检测医疗器械的细菌和真菌污染。但该测试方法存在以下缺点： 鲎试验无法检测肽聚糖；由于脂多糖和β-1,3-葡聚糖检测呈阳性，无法区分检测到的污染是细菌还是真菌造成的；鲎试验采用的是鲎的细胞裂解液，未来可能会出现鲎供应不足的情况。本研究项目的目的是开发一种市售试剂，专门检测微生物细胞壁成分。蚕 (Bombyx mori) 血浆含有由肽聚糖和 β-1, 3-葡聚糖触发的原酚氧化酶级联。我们打算使用级联来检测细菌和真菌对医疗器械的污染。我们成功开发了一种定量肽聚糖或 β-1, 3-葡聚糖的方法，其浓度非常低，例如几 pg/ml。此外，我们可以通过使用针对β-1, 3-葡聚糖识别蛋白的抗体，使家蚕原酚氧化酶级联对肽聚糖具有特异性，β-1, 3-葡聚糖识别蛋白是原酚氧化酶级联的一个组成部分，并与β-1, 3-葡聚糖特异性相互作用。这些对酚氧化酶原级联的研究结果表明，该级联是检测和定量医疗器械微生物污染的试剂的良好候选者，特别是革兰氏阳性菌的检测。我们正在开发方法来收集大量未触发级联的蚕血浆，并稳定血浆中的级联，以便将血浆从供应商运输到用户。

项目成果

Brey P. T., Stoltz, D. B., Cook, D. I. and Ashida, M.: "Use of nitrocellulose membrane to activate and measure insect phenoloxidase." Anal. Biochem.

Brey P. T.、Stoltz, D. B.、Cook, D. I. 和 Ashida, M.：“使用硝化纤维素膜激活和测量昆虫酚氧化酶。”

Ochiai M. and Ashida M.: "Purification of a beta-1, 3-glucan recognition protein in the prophenoloxidase activating system from hemolymph of the silkworm, Bombyx mori." J. Biol Chem.263. 12056-12062 (1988)

Ochiai M. 和 Ashida M.：“从家蚕血淋巴中纯化酚氧化酶原激活系统中的 β-1, 3-葡聚糖识别蛋白。”

Ochiai,M.: J.Biol.Chem.263. 12056-12062 (1988)

落合，M.：J.Biol.Chem.263。

Ashida M. and Yoshida H.: "Limited proteolysis of prophenoloxidase during activation by microbial products in insect plasma and effect of phenoloxidase on electrophoretic mobilities of plasma proteins." Insect Biochem. 18. 11-19 (1988)

Ashida M. 和 Yoshida H.：“昆虫血浆中微生物产物激活过程中酚氧化酶原的有限蛋白水解以及酚氧化酶对血浆蛋白电泳迁移率的影响。”

Ashida M., Ochiai M. and Niki, T.: "Immunolocalization of prophenoloxidase among hemocytes of the silkworm, Bombyx mori" Tissue & Cell. 20. 599-610 (1988)

Ashida M.、Ochiai M. 和 Niki, T.：“蚕 Bombyx mori 血细胞中酚氧化酶原的免疫定位”组织