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.

本项目的最终目的是开发一种检测和定量微量肽聚糖和β-1，3-葡聚糖的方法。虽然已经通过使用鲎（Limulus）的血细胞裂解物上清液开发了用于脂多糖和β-1，3-葡聚糖的这种方法，但是通过该方法不能检测肽聚糖。先前我们报道家蚕血淋巴的血浆部分含有由微量肽聚糖或β-1，3-葡聚糖触发的原酚氧化酶级联反应。研究了血浆组分是否可用于检测和定量肽聚糖，以及是否可在其性质变化最小的情况下进行商业供应。为进一步提高该方法的可行性，本文还对肽聚糖和β-1，3-葡聚糖激活多酚氧化酶级联反应的机理进行了研究。1）建立了家蚕血淋巴的处理方法，为肽聚糖和β-1，3-葡聚糖的检测和定量提供血浆组分。2）SLP-试剂的酚氧化酶级联反应可由肽聚糖和β-1，3-葡聚糖两者触发。由肽聚糖或β-1，3-葡聚糖特异性触发的SLP试剂是理想的。我们开发了一种方法，使SLP试剂特异性肽聚糖或β-1，3-葡聚糖。这种提高家蚕血浆特异性的方法已有报道，但其缺点是血浆中的酚氧化酶级联反应在血浆操作过程中启动。新开发的方法降低了自发激活的风险。目前，我们成功地获得了对肽聚糖激活的原酚氧化酶级联反应敏感10，000倍的家蚕血浆。

项目成果

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。

Ashida,M.: "Role of the integument in insect defense : Prophenoloxidase cascade in the cuticular matrix" Proc.Natl.Acad.Sci.U.S.A.92. 10698-10702 (1995)

Ashida,M.：“体被在昆虫防御中的作用：角质层基质中的酚氧化酶级联”Proc.Natl.Acad.Sci.U.S.A.92。

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。

Yasuhara,Y.: "Re-examination of properties of prophenoloxidase isolated from larval hemolymph of the silkworm,Bombyx mori" Arch.Biochem.Biophys.320. 14-23 (1995)

Yasuhara，Y.：“从蚕幼虫血淋巴中分离出的酚氧化酶原的特性的重新检查，家蚕”Arch.Biochem.Biophys.320。

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。”