The development of a method for detection and quantification of cell wall components of bacteria and fungi.

开发一种检测和定量细菌和真菌细胞壁成分的方法。

• 批准号: 06554037
• 负责人: ASHIDA Masaaki
• 金额: $ 13.18万
• 依托单位: HOKKAIDO UNIVERSITY
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (A)
• 财政年份: 1994
• 资助国家: 日本
• 起止时间: 1994 至 1996
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-06554037/
• 关键词: Peptidoglycan; beta-1,3-Glucan; Phenol oxidase; cascade; Hemolymph; Lipopolysaccharide; Limulus test; SLP-reagent; 昆虫; フェノールオキシダーゼ; 汚染検査; プロテアーゼ; β-1,3-グルカン; 昆虫血液; β-1、3-グルカン; 細菌; カビ; リムルス

The end of the present project is to develop a method to detect and quantify minute amounts of peptidoglycan and beta-1,3-glucan. Although such method for lipopolysaccharide and beta-1,3-glucan has been developed by using hemocyte lysate supernatant of horseshoe crab (Limulus), peptidoglycan could not be detected by the method. Previously we reported that plasma fraction of silkworm hemolymph contains prophenol oxidase cascade that is triggered by minute amount of peptidoglycan or beta-1,3-glucan. The plasma fraction was studied if it could be used for detection and quantification of peptidoglycan and if it could be supplied commercially with minimal variation of its property. Toimprove the feasibility of the method, the mechanism of the activation of prophenol oxidase cascade with peptidoglycan or beta-1,3-glucan was also studied. The major results obtained during the term of the present project are as follows :1) A method for processing silkworm hemolymph was developed for supplying plasma fraction for the detection and quantification of peptidoglycan and beta-1,3-glucan. The plasma fraction is now available commercially under a name of SLP-reagent.2) Prophenol oxidase cascade of SLP-reagent could be triggered by bothof peptidoglycan and beta-1,3-glucan. SLP-reagent which is specifically triggered by peptidoglycan or by beta-1,3-glucan is desirable. We developed a method to make SLP-reagent specific to peptidoglycan or beta-1,3-glucan. This kind of method for raising the specificity of silkworm plasma has already been reported, but it had weak point in that prophenol oxidase cascade in plasma get start of activation during the manipulation of plasma. The newly developed method diminished the risk for the spontaneous activation. At present we succeeded in obtaining silkworm plasma of which prophenol oxidase cascade is 10,000 times more sensitive for being activated by peptidoglycan.

本项目的终结是开发一种检测和量化微小量的肽聚糖和β-1,3-葡聚糖的方法。尽管通过使用马蹄蟹（Limulus）的血细胞裂解物上清液（Limulus），已经开发了这种用于脂多糖和β-1,3-葡聚糖的方法，但该方法无法检测到肽聚糖。以前，我们报道了蚕血淋巴的等离子体分数含有预知氧化酶级联反应，这些级联由微量肽聚糖或β-1,3-葡聚糖触发。如果可以将其用于检测和定量肽聚糖，并且可以在商业上提供其特性变化的最小变化，则研究了该血浆分数。为了解脱该方法的可行性，还研究了先知氧化酶级联反应的机制，它还研究了肽聚糖或β-1,3-葡聚糖。在本项目期间获得的主要结果如下：1）开发了一种用于供应等离子分数来检测和定量肽聚糖和β-1,3-葡聚糖的方法。现在以Slp-ragent的名称在商业上获得血浆分数。2）SLP-REATENT的先知氧化酶级联反应可能是由肽聚糖和β-1,3-葡聚糖均可触发的。 SLP反应是由肽聚糖或β-1,3-葡聚糖特别触发的。我们开发了一种方法，使SLP更适合于肽聚糖或β-1,3-葡聚糖。已经报道了提高蚕血浆特异性的这种方法，但在血浆操纵过程中，血浆中的先知氧化酶级联反应较弱。新开发的方法减少了自发激活的风险。目前，我们成功地获得了蚕血浆，其中预知氧化酶级联对被肽聚糖激活的敏感性高10,000倍。

项目成果

Yoshida, H.: "Purification of a peptidoglycan recognition protein from hemolymph of the silkworm, Bombyx mori" J. Biol. Chem.271. 13854-13860 (1996)

Yoshida, H.：“从蚕（Bombyx mori）血淋巴中纯化肽聚糖识别蛋白”J. Biol。

Lee, W.-J.: "Molecular cloning and chromosomal localization of prophenoloxidase cDNA from the malaria vector Anopheles gambiae." Biochem.J.(in press). (1996)

Lee, W.-J.：“来自疟疾载体冈比亚按蚊的酚氧化酶原 cDNA 的分子克隆和染色体定位。”

Brey,P.T.: "Tyrosinase-type prophenoloxidase distribution in the alimentary canal of strains of Anopheles gambiae refractory and susceptible to Plasmodium infection" Experimental Parasitol.80. 654-664 (1995)

Brey，P.T.：“酪氨酸酶型酚氧化酶原在难治且易受疟原虫感染的冈比亚按蚊菌株消化道中的分布”实验性寄生虫醇.80。

Ashida, M.: "Recent advances on the research of the insect prophenoloxidase cascade" Molecular mechanisms of immune responses insects. (印刷中). (1997)

Ashida, M.：“昆虫原酚氧化酶级联研究的最新进展”昆虫免疫反应的分子机制（正在出版）。

Y.Katsumi et al.: "A serine protease zymogen in insect plasma:Purification and activation by microbial cell wall components" Eur.J.Biochem.(in press). (1995)

Y.Katsumi 等人：“昆虫血浆中的丝氨酸蛋白酶原：微生物细胞壁成分的纯化和活化”Eur.J.Biochem.（出版中）。