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.

应用噬菌体载体L47.1体外包装技术构建了变形链球菌B13 N（d型）染色体DNA基因库<lambda><lambda>。将噬菌体基因库感染E.大肠杆菌中分离纯化的葡糖基转移酶（glucosyltransferase synthesizing insoluble glucan，I-GTF）的兔抗血清，通过免疫印迹ELISA法筛选I-GTF克隆。变异人有几个克隆具有蔗糖水解活性。进一步<lambda>研究了命名为MDSM 39的一个克隆。通过<lambda>SDS-PAGE和HPLC凝胶过滤分析，来自MDSM 39的I-GTF具有约160 kdal的分子量，并且显示出约3.8的PI。这种I-GTF活性通过添加可溶性葡聚糖合成的GTF或葡聚糖T10来刺激。MDSM 39合成的不溶性葡聚糖<lambda>主要由<alpha>-1，3糖苷键组成。这些数据表明S.成功克隆了变形杆菌I-GTF基因，并在E.杆菌<lambda>MDSM 39克隆不仅可用于酶学和分子生物学研究，而且可用于防龋疫苗的研制。

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。