Gene Cloning of Oral Streptococcal Glucanases

口腔链球菌葡聚糖酶的基因克隆

基本信息

批准号：
60570859
负责人：
FUKUSHIMA Kazuo
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-60570859/
关键词：
Gene Cloning Dental Plaque Mutanase Dextranase 口腔レンサ球菌

项目摘要

Cariogenic bacterium Streptococcus mutans synthesizes extracellularly adhesive water-insoluble glucans (mutan, ad-WIG) from sucrose by the combined action of two or three types of glucosyltransferase (GTase), resulting in the adherence of cells to smooth surfaces and cariogenic plaque formation. There are many experimental evidences with respect to the inhibition of ad-WIG and plaque formation by glucanases. Thus, in order to realize the mass production of glucanases and the gene expression in oral cavity, the cloning of glucanase genes from oral streptococci was carried out in this investigation.Ad-WIG was enzymatically synthesized using GTases purified from S mutans B13-N, and <alpha> -1,3 glucan was prepared by chemical treatments of ad-WIG. Mutanase-producing bacterium (turmed MU-strain) was isolated from human oral-fluids, by the use of BHI agar plates supplemented with ad-WIG. This isolate was identified as Streptococcus mitior, from its morphological and biochemical characteristics. The MU-strain produced extracellulerly dextranase, in addition to intracelluler mutanase. Both glucanases were partially purified and used as antigens for preparation of the antiserums. From the mutanolysin-treated cells, high-moleculer-weight (>20 Kb) chromosomal DNA was prepared, digested with sau3AI, ligated with BamHI fragments of <lambda> L47.1 DNA, and in vitro packaging was done with a GIGA pack system. Consequently, the usefull clone bank was obtained. The clone bank was screened for glucanase clones by an immunobloting method using a mixture of anti-dextranase and anti-mutanase serums. Seven phage clones stained by peroxidase were isolated. But, none of the antigen-positive clones expressed any glucanase activity, when the phage lyzates were incubated at 37゜C for 48 h with <alpha> -1,3 glucan or dextran T-2000.

商菌细菌链球菌链球菌通过两种或三种类型的葡萄糖基转移酶（GTase）的联合作用从蔗糖中合成细胞外粘合性水独立的葡萄糖（Mutan，Ad-Wig），从而导致细胞的粘附以平滑表面和石化的质膜形成。关于葡萄糖酶抑制AD-WIG和斑块形成的实验证据。为了实现葡萄糖酶的质量产生和口腔中的基因表达，在这项投资中进行了口服链球菌的克隆基因的克隆。AD-WIG使用从S Mutans B13-N纯化的GTase合成AD-WIG，并通过B13-N，<Alpha> -1-1,3 glucan制备了Ad-wig的Ad-Wig。通过使用补充AD-WIG的BHI琼脂平板，从人的口腔液体中分离出产生型糖酶的细菌（turmed Mu-strain）。从其形态和生化特征中鉴定为链球菌。除尿素内突变酶外，MU-STRAIN还产生了外尿促右旋酶。两种葡萄糖酶均部分纯化，并用作抗原的抗原。从毒素蛋白蛋白处理的细胞中，制备了高分子量（> 20 kb）染色体DNA，并用sau3ai消化，并用<lambda> l47.1 DNA的Bamhi片段结扎，并使用GIGA包装系统完成了体外包装。因此，获得了有用的克隆银行。通过使用抗脱去酶和抗糊状酶血清的混合物，通过一种免疫印迹方法对克隆库进行麸烷酶克隆筛选。分离了通过过氧化物酶染色的七个噬菌体克隆。但是，当将噬菌体在37°C下与<Alpha> -1,3葡萄糖或葡萄糖T-2000孵育48小时时，均无抗原阳性克隆表达任何谷氨酸酶活性。