Gene Cloning of Oral Streptococcal Glucanases

链球菌突变链球菌综合了提取物附着的水不溶葡萄糖（mutan， ad-WIG），由两种或三种类型的葡萄糖基转移酶（GTase）的结合作用，在细胞对光滑表面和钙基蛋白形成的优势中的结果。有许多实验证据，对Glucanases的Ad-WIG和Plaque形成的影响表示赞赏。因此，为了确定葡萄糖的质量生产和口腔洞穴中的基因表达，葡萄糖基因的克隆从口腔链球菌中携带出来，Ad-WIG利用Smutans B13-N和<alpha>-1，3葡萄糖是由Ad-WIG的化学处理准备的。Mutanase生产的细菌（turmed MU-strain）从人类口腔流体中分离出来，由BHI琼脂板补充而成。这种孤立物被识别为链球菌，来自其形态学和生物化学特征学。MU-strain产生的提取物脱氧酶，在添加到Intracellulerly突变酶。这两种葡萄糖都是部分纯化的，并被用作用于准备抗血清的抗原。来自经突变体治疗的细胞，高分子重量（&gt;20 Kb）的染色体DNA准备，用Sau 3AI进行挖掘，<lambda>用L47.1 DNA的BamHI片段进行了DNA鉴定，用GIGA包装系统进行了体外包装。顺便说一下，现有的克隆银行被发现了。克隆库被筛选为葡萄糖酶克隆体，使用一种免疫印迹方法来混合反脱氨酶和反突变酶血清。七个阶段克隆被过氧化物酶孤立化。但是，没有抗基因阳性克隆人表现出任何葡萄糖酶活性，当相位溶解在37 ° C时孵化为48小时，与<alpha>-1，3葡萄糖或右旋糖酐T-2000。

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.