Gene Cloning of Glucosyltransferase Synthesizing Insoluble Glucan from Streptococcus mutans

以噬菌体载体&lt；lambda&gt；L47.1为载体，采用体外包装技术构建了变形链球菌B13N(血清型d)染色体DNA基因库。将噬菌体基因库导入大肠杆菌，用兔抗变形链球菌葡聚糖合成不溶性葡聚糖(I-GTF)的抗血清，通过免疫印迹ELISA法筛选出I-GTF克隆。几个无性系具有蔗糖水解酶活性。对一个命名为&lt；lambda&gt；MDSM39的克隆进行了进一步研究。经SDS-PAGE和高效液相凝胶过滤分析，MDSM39的I-GTF的相对分子质量约为160kdal，等电点约为3.8。这种I-GTF活性可被合成的可溶性葡聚糖或葡聚糖T10所刺激。~lt；lambda&gt；MDSM39合成的不溶性葡聚糖的组成主要是-lt；α&gt；-1，3-葡萄糖苷键。这些结果表明，变形链球菌I-GTF基因已成功克隆并在大肠杆菌中进行了功能表达。MDSM39克隆将是一种有用的材料，不仅用于酶和分子生物学研究，而且还用于研制龋病疫苗。

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.