Characterization of regular array protein in the cell wall of Lactobacillus

乳酸菌细胞壁规则阵列蛋白的表征

• 批准号: 63570195
• 负责人: KOGA Tetsuro
• 金额: $ 1.34万
• 依托单位: TOKUSHIMA UNIVERSITY
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for General Scientific Research (C)
• 财政年份: 1988
• 资助国家: 日本
• 起止时间: 1988 至 1989
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-63570195/
• 关键词: Lactobacillus; Regular array; Gene library; Immunoscreening; 免疫ブロット

1. Lactobacillus acidophilus was composed of many bacterial strains with very heterogeneous biological and biochemical characteristics. We showed possibility that in addition to classification by DNA-DNA homology,character of regular array protein of the organism could be used to classify L. acidophilus strains. Only the strains of DNA homology groupA produced RA proteins and no other groups produced. All A-1 subgroup strains produced RA proteins with same molecular weight and peptidemapping. Furthermore, they showed high homology between their immunological properties. In contrast, RA proteins of subgroup A-2, A-3 and A-4 were differed from each other in their molecular weight and peptide mapping. Although, immunological properties of subgroup A-2 RA proteins showed considerably homogeneous, those of subgroup A-3 and A-4 strains appeared heterogeneous. We conclude that there are necessity to classify subgroup A-3 and A-4 by means of properties of RA proteins.2. We tried to clone the regular array protein gene of L. brevis ATCC8287. The chromosomal DNA fragments(3-9 kb) produced bypartial digestion with EcoRI was ligated with gt 11 EcoRI digested DNA and then packaged in vitro. Sixteen positive clones were screened from 2638 plaques by immunoscreening with anti-RA protein antibody. When we examined the lysate of ten positive clones by Western blotting, seven clones produced immunopositive protein with molecular weight more than 130 kDa, and three clones made positive protein with a molecular weight lower than 130 kDa. The former phage DNA contained 2.4 kb EcoRI fragment and the latter contained 3.2 kb EcoRI fragment. The 2.4 kb EcoRI fragment was cloned in the plasmid pU19 to construct pLB1, which was expressed in E.coli JM109 and produced the RA protein identical in molecular weight to the purified RS protein. The delated mutant of pLB1 was produced and work is in progress to sequence the 2.4 kb fragment of the cloned DNA.

1。乳杆菌由许多细菌菌株组成，具有非常异质的生物学和生化特征。我们表明，除了通过DNA-DNA同源性分类外，生物体的常规阵列蛋白质的特征还可以用于对嗜酸乳杆菌菌株进行分类。只有DNA同源性群体的菌株会产生RA蛋白，而没有产生其他组。所有A-1亚组菌株均产生具有相同分子量和肽膜的RA蛋白。此外，它们在免疫学特性之间表现出很高的同源性。相反，亚组A-2，A-3和A-4的RA蛋白在其分子量和肽映射方面彼此不同。尽管亚组A-2 RA蛋白的免疫学特性表现出相当均匀的，但亚组A-3和A-4菌株的免疫学特性似乎异质。我们得出的结论是，有必要通过RA蛋白质的特性对亚组A-3和A-4进行分类。2。我们试图克隆Brevis Atcc8287的常规阵列蛋白基因。用gt 11 Ecori消化的DNA结扎了子宫菌消化的染色体DNA片段（3-9 kb），然后在体外包装。通过用抗RA蛋白抗体免疫统计，从2638颗斑块中筛选了16个阳性克隆。当我们通过蛋白质印迹检查了十个阳性克隆的裂解物时，七个克隆产生了分子量超过130 kDa的免疫阳性蛋白，而三个克隆产生了阳性蛋白，分子量低于130 kDa。以前的噬菌体DNA包含2.4 kb的Ecori片段，后者包含3.2 kb Ecori片段。将2.4 kb的ECORI片段克隆在质粒PU19中以构建PLB1，该PLB1在E.Coli JM109中表达，并产生与纯化RS蛋白的分子量相同的RA蛋白。产生了PLB1的稀释突变体，并且正在进行工作以对克隆DNA的2.4 kb片段进行测序。

项目成果

古賀哲郎: "Lacto bacillus acidophilus細胞壁の規則的配列構造蛋白質刃の遺伝子解析" 日本細菌学誌. 45. 452 (1990)

Tetsuro Koga：“嗜酸乳杆菌细胞壁规则排列的结构蛋白叶片的遗传分析”日本细菌学杂志 45. 452 (1990)。

古賀哲郎: "Lactobacillus acidophilus諸菌株の細胞壁規則的配列構造蛋白質の単離と免疫化学的性状および分類学的意義" 日本細菌学雑誌. 44. 320 (1989)

Tetsuro Koga：“嗜酸乳杆菌菌株规则排列的细胞壁结构蛋白的分离、免疫化学特性和分类学意义”《日本细菌学杂志》44. 320 (1989)。

Tetsuro Koga: "Cloning of the cell wall regular array protein gene of Lactobacillus brevis" Jpn. J. Bacteriol., 45 (1), 452 (1990).

Tetsuro Koga：“短乳杆菌细胞壁规则阵列蛋白基因的克隆”Jpn。

川田十三夫: "Lactobacillus acidophilusにおける細胞壁の規則的配列構造の分布、性状および分類学的意義" 乳酸菌研究会に関する報告書(昭和62年度). 332-335 (1988)

Juzo Kawata：“嗜酸乳杆菌细胞壁规则排列结构的分布、特性和分类学意义”乳酸菌研究组报告 (1988) (332-335)。

古賀哲郎: 日本細菌学雑誌. 44. 320 (1989)

Tetsuro Koga：日本细菌学杂志 44. 320 (1989)。