Gene Cloning of Glucosyltransferase Synthesizing Insoluble Glucan from Streptococcus mutans

变形链球菌合成不溶性葡聚糖的葡萄糖基转移酶基因克隆

• 批准号: 60570881
• 负责人: ABIKO Yoshimitsu
• 金额: $ 1.09万
• 依托单位: Nihon 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: 1985
• 资助国家: 日本
• 起止时间: 1985 至 1986
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-60570881/
• 关键词: Gene Cloning; Streptococcus mutans; Escherichia coli; Glucosyltransferase; Insoluble Glucan

Gene banks of Streptococcus mutans B13N (serotype d) chromosomanl DNA in Escherichia coli were constructed by phage vector <lambda> L47.1 using <lambda> in vitro packaging technique. Phage gene banks were infected into E. coli, and glucosyltransferase synthesizing insoluble glucan (I-GTF) clones were screened by immuno-blot ELISA procedure using rabbit antiserum against I-GTF purified from S. mutans. Several clones had sucrose hydrolyzing activities. One clone designated as <lambda> MDSM39 was further studied. The I-GTF from <lambda> MDSM39 had molecular weight approximately 160 kdal by SDS-PAGE and HPLC gel filtration analysis, and exhibited a PI of approximately 3.8. This I-GTF activity was stimulated by the addition of soluble glucan synthesized GTF or dextran T10. The composition of insoluble glucan synthesized by <lambda> MDSM39 was mainly <alpha> -1,3 glucoside linkage. These data suggested that S. mutans I-GTF gene was successfully cloned and functionally expressed in E. coli. <lambda> MDSM39 clone will be a usefull material not only for enzymological and molecularbiological studies but also for development of vaccination against dental caries.

利用噬菌体载体<lambda>L47.1，采用体外包装技术，构建了大肠杆菌变形链球菌B13N（血清型d）染色体DNA基因库。将噬菌体基因库感染到大肠杆菌中，并使用从变形链球菌中纯化的针对 I-GTF 的兔抗血清，通过免疫印迹 ELISA 程序筛选合成不溶性葡聚糖 (I-GTF) 的葡糖基转移酶克隆。几个克隆具有蔗糖水解活性。进一步研究了命名为MDSM39的一个克隆。通过SDS-PAGE和HPLC凝胶过滤分析，来自λMDSM39的I-GTF具有大约160kdal的分子量，并且表现出大约3.8的PI。通过添加可溶性葡聚糖合成的 GTF 或葡聚糖 T10 可以刺激 I-GTF 活性。由<lambda>MDSM39合成的不溶性葡聚糖的组成主要是<α>-1,3葡萄糖苷键。这些数据表明变形链球菌I-GTF基因已成功克隆并在大肠杆菌中功能表达。 MDSM39克隆不仅对于酶学和分子生物学研究而且对于开发针对龋齿的疫苗接种都是有用的材料。

项目成果

Yoshimitsu Abiko;Hisashi Takiguchi: J.Dental Research. 64. 737 (1985)

Yoshimitsu Abiko；Hisashi Takiguchi：J.Dental Research。

Yoshimitsu Abiko and Hisashi Takiguchi: "Cloning and Expression of Glucosyltransferase Gene from Streptococcus mutans B13 in Escherichia coli" J. Dental Research. 64. 737 (1985)

Yoshimitsu Abiko 和 Hisashi Takiguchi：“变形链球菌 B13 的葡萄糖基转移酶基因在大肠杆菌中的克隆和表达”J. Dental Research。

Yoshimitsu Abiko, Mitsuo Hayakawa, Hidefumi Aoki, Kazuo Fukushima and Hisashi Takiguchi: "Molecular Cloning of Glucosyltransferase Synthesizing Insoluble Glucan from Streptococcus mutans B13N" FEBS Letters.

Yoshimitsu Abiko、Mitsuo Hayakawa、Hidefumi Aoki、Kazuo Fukushima 和 Hisashi Takiguchi：“从变形链球菌 B13N 合成不溶性葡聚糖的葡萄糖基转移酶的分子克隆”FEBS 快报。

Yoshimitsu Abiko;Mitsuo Hayakawa;Hidefumi Aoki;Kazuo Fukushima;Hisashi Takiguchi: FEBS Letters.

安子子义光、早川光夫、青木英文、福岛一夫、泷口尚：FEBS 信件。