Gene Cloning of Glucosyltransferase Synthesizing Insoluble Glucan from Streptococcus mutans

采用<lambda>体外包装技术，以噬菌体载体<lambda> L47.1构建大肠杆菌突变链球菌（d血清型）染色体DNA基因库。将噬菌体基因库感染大肠杆菌，利用从变形链球菌中纯化的I-GTF兔抗血清，采用免疫印迹ELISA法筛选糖基转移酶合成不溶性葡聚糖（I-GTF）克隆。几个无性系具有蔗糖水解活性。对其中一个命名为<lambda> MDSM39的克隆进行了进一步研究。经SDS-PAGE和HPLC凝胶过滤分析，<lambda> MDSM39的I-GTF分子量约为160 kdal， PI约为3.8。可溶葡聚糖合成GTF或葡聚糖T10可刺激I-GTF活性。< λ > MDSM39合成的不溶性葡聚糖的组成主要是< α > -1,3糖苷链。这些数据表明，突变链球菌I-GTF基因已成功克隆并在大肠杆菌中功能表达。MDSM39克隆不仅可用于酶学和分子生物学研究，而且可用于开发抗龋疫苗。

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.