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本文研究高粱外种皮原花青素四聚体(sorghum episperm procyanidin tetramer,SEPT)对远缘链球菌6715(S.sobrinus 6715)初始粘附及其致龋毒力因子-葡萄糖基转移酶(glucosyltransferase,GTF)的抑制效果。采用唾液包被羟基磷灰石(saliva coated hydroxylapatite,SHA)体外模型研究SEPT对S.sobrinus 6715初始粘附的影响。通过PCR扩增S.sobrinus 6715 GTF-I的催化活性区(catalytic region,CAT)的基因片段到质粒Pgex-4T-1中,转化至大肠杆菌T7 Express competent,IPTG诱导表达,纯化浓缩表达产物,获得目标蛋白GTF-I/CAT。再用不同浓度SEPT处理GTF-I/CAT,根据其水不溶性多糖产量(water insoluble glucosyltransferase,WIS)评价SEPT对GTF-I/CAT酶活力的抑制。实验结果表明,SEPT能够有效抑制S.sobrinus 6715在SHA的初始粘附,并具有一定的浓度效应关系;通过基因克隆表达获得的GTF-I/CAT蛋白酶活为8.446 m IU,产多糖能力为1.603 mg/(mg·h)。SEPT能明显抑制GTF-I/CAT产生WIS(p<0.01)。说明SEPT既可以有效抑制S.sobrinus 6715的初始粘附过程,又可以通过作用GTF-I的CAT区减少WIG的合成,抑制蔗糖依赖性粘附减少S.sobrinus 6715在牙齿表面的粘附聚集,从而干预龋齿的发生。
In this paper, we investigated the initial adhesion of sorghum episperm procyanidin tetramer (SEPT) to S.sobrinus 6715 and its cariogenic virulence factor - glucosyltransferase (GTF) Inhibitory effect. Saliva coated hydroxyapatite (saliva coated hydroxylapatite, SHA) in vitro model of SEPT on S. sobrinus 6715 initial adhesion. The gene fragment of the catalytic region (CAT) of S. sobrinus 6715 GTF-I was amplified by PCR into plasmid pgex-4T-1 and transformed into E. coli T7 Express competent. The expression product was induced by IPTG, Get the target protein GTF-I / CAT. GTF-I / CAT was treated with different concentrations of SEPT, and the inhibition of GTF-I / CAT activity was evaluated based on its water insoluble glucosyltransferase (WIS). The experimental results showed that SEPT could effectively inhibit the initial adhesion of S.sobrinus 6715 to SHA and had a concentration-dependent effect. The GTF-I / CAT protease activity obtained by gene cloning was 8.446 m IU and the polysaccharide productivity was 1.603 mg / (mg · h). SEPT significantly inhibited Wistar response to GTF-I / CAT (p <0.01). SEPT can inhibit the initial adhesion process of S.sobrinus 6715 and reduce the synthesis of WIG by acting on the CAT region of GTF-I, inhibiting the sucrose-dependent adhesion and reducing the adhesion and aggregation of S.sobrinus 6715 on the tooth surface, In order to interfere with the occurrence of dental caries.