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

摘要:

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 mIU，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。

Abstract:

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 mIU 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.