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Research on complex bactericidal compounds in the hemolymph of silkworms

家蚕血淋巴中复杂杀菌化合物的研究

基本信息

批准号：
02453027
负责人：
MURAE Tatsushi
金额：
$ 4.67万
依托单位：
Kyushu University (1991)The University of Tokyo (1990)
依托单位国家：
日本
项目类别：
Grant-in-Aid for General Scientific Research (B)
财政年份：
1990
资助国家：
日本
起止时间：
1990 至 1991
项目状态：
已结题

项目摘要

Hemolymph from 3, 000 fifth-instar silkworms was pooled, frozen and stored at -80ﾟC. Escherichia coli B/SM, a spontaneously occurring mutant strain resistant to streptomycin, was used for the estimation of bactericidal activity.By affinity chromatography using crab shell chitin powder, the hemolymph afforded a path through fraction (fraction A : eluted with a neutral buffer solution) and a lysozyme-like fraction (eluted with 0.1N acetic acid). Both fractions lysed Micrococcus Lysodeikticus. The lysozyme-like fraction alone could kill E. coli.The lysozyme-like fraction was further fractionated by a gel-filtration chromatography (GFC). The major fractions which killed E. coli on combination with the fraction A exhibited the bactericidal activity without the fraction A. The active fractions were combined and fractionated by a cation exchange chromatography to yield three lysozyme-like components, two of which killed E. coli without cofactors. The bactericidal activity of the major component, which was eluted from the cation exchange chromatography earliest among the three lysozyme-like fractions, was remarkably enhanced by addition of the fraction A. An electrophoresis of the major bactericidal component showed a single band having more basicity than egg-white lysozyme.Among the fractions obtained by GFC of the fraction A, there were three types of bactericidal components : 1) killed E. coli without lysozyme-like factors, and the addition of the lysozyme-like fraction did not seriously affect the activity, 2) killed E. coli without lysozyme-like factors and the addition of the lysozyme-like fraction diminished the activity, and 3) killed E. coli by combination with the lysozyme-like factors and the addition of the lysozyme-like fraction enhanced the activity remarkably. These facts indicate the presence of very complex mechanisms of selfdefense in the normal, cell-free hemolymph of silkworms.

将来自3000只五龄蚕的血淋巴合并，冷冻并储存在-80 ℃下。大肠杆菌（Escherichia coli）B/SM是一株自然产生的链霉素抗性突变株，其血淋巴经蟹壳几丁质粉末亲和层析，得到一个通过组分（组分A：用中性缓冲液洗脱）和一个溶菌酶样组分（用0.1N乙酸洗脱）的通道。两种级分均裂解溶壁微球菌。溶菌酶样组分本身就能杀死大肠杆菌。通过凝胶过滤层析（GFC）进一步分离溶菌酶样组分。杀死大肠杆菌的主要组分。大肠杆菌与级分A组合时，在没有级分A的情况下表现出杀菌活性。将活性组分合并，通过阳离子交换层析分离，得到三种溶菌酶样组分，其中两种杀死E。不含辅因子的大肠杆菌。在三种溶菌酶样组分中，最早从阳离子交换色谱中洗脱的主要组分的杀菌活性通过加入组分A而显著增强。主要杀菌成分的电泳显示一条碱性强于蛋清溶菌酶的单一条带，在GFC得到的组分A中，有3种杀菌成分：1）杀灭E.大肠杆菌中的溶菌酶样因子，而溶菌酶样因子的加入并不严重影响其活性;大肠杆菌中的溶菌酶样因子和溶菌酶样组分的加入降低了活性;大肠杆菌的溶菌酶样因子的组合和溶菌酶样组分的加入显着提高了活性。这些事实表明，在正常的家蚕无细胞血淋巴中存在非常复杂的自卫机制。