高粱外种皮原花青素四聚体对远缘链球菌6715粘附的影响

Effect of Procyanidin Tetramer from Sorghum Episperm on the Adherence of Streptococcus sobrinus 6715

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。

The inhibitory effect of sorghum episperm procyanidin tetramer（SEPT） on the initial adherence of Streptococcus sobrinus 6715 and its cariogenic factor glucosyltransferase（GTF） was investigated.The saliva-coated hydroxyapatite（SHA） was used as an in vitro model to measure the inhibitory effect of SEPT on the initial adherence of S.sobrinus 6715.The GTF-I catalytic region（CAT） gene of S.sobrinus 6715 was amplified by polymerase chain reaction（PCR）,cloned into a Pgex-4T-1 vector,and transformed into T7 Express competent of Escherichia coli.The protein expression was induced by isopropyl β-D-1-thiogalactopyranoside（IPTG）,and the expressed product was purified and concentrated to obtain the GTF-I/CAT target protein.Subsequently,different concentrations of SEPT samples were used to treat GTF-I/CAT,and the inhibitory effect of SEPT on GTF-I/CAT enzyme activity was determined based on the amount of water-insoluble glucan（WIG） formed by GTF-I/CAT.The results showed that SEPT could effectively decrease the initial adherence of S.sobrinus 6715 to SHA in a dose-dependent manner.The enzyme activity of GTF-I/CAT obtained from the gene cloning and expression was 8.446 m IU and the WIG formation ability was 1.603 mg/（mg·h）.SEPT could effectively suppress the formation of WIG by acting on GTF-I/CAT（P〈0.01）.The results demonstrated that SEPT was effective in inhibiting the initial adherence of S.sobrinus 6715 to SHA and reducing WIG synthesis by acting on the CAT region of GTF-I.Therefore,SEPT can decrease the adhesion and aggregation of S.sobrinus 6715 on tooth surface and inhibit the sucrose-dependent adherence,thereby preventing the occurrence of dental caries.