在大肠杆菌中表达和提取纯化变形链球菌GTF—I

葡糖基转移酶是变形链球菌 (Ｓ ｍｕｔａｎｓ)的重要致龋毒力因子 ,其中ｇｔｆＢ基因编码的ＧＴＦ Ⅰ能够催化水不溶性葡聚糖的合成 ,介导变链菌与牙面的蔗糖依赖性粘附 ,在龋病的发生和发展中起重要作用。目前我们已研制了针对变链菌的表面蛋白ＰＡｃ和ＧＴＦ的融合防龋ＤＮＡ疫苗[1 ] ,为了进一步鉴定和加强其免疫效果 ,我们以金属螯合亲和层析法从大肠杆菌中表达、提取和纯化了重组葡糖基转移酶ＧＴＦ Ⅰ。一、材料和方法1 .重组葡糖基转移酶ＧＴＦ Ⅰ表达质粒的构建 :根据Ｓ ｍｕｔａｎｓＧＳ 5株葡糖基转移酶基因ｇｔｆＢ的序列设计扩…     

唾液对变形链球菌Mutans和变形链球菌Sobrinus粘附的影响

用＾３Ｈ标记变形链球菌遗传分型Ⅰ型Ｓ．ｍｕｔａｎｓＪＢＰ和ⅢＳ．ｓｏｂｒｉｎｕｓ６７１５，观察细菌在经兔唾液包被的羟磷灰石微珠表面的粘附量，以研究唾液对两种菌粘附的影响。结果表明，Ｓ．ｍｕｔａｎｓＪＢＰ和Ｓ．ｓｏｂｒｉｎｕｓ６７１５对经唾液包被的ＨＡ上的粘附量有明显差异，Ｉ型ＪＢＰ的粘附量显著多于Ⅲ型６７１５，在无唾液包被的ＨＡ上，两者粘附量无明显差异。结果说明变形链球菌Ｉ型Ｓ．ｍｕｔａｎｓ粘附的     

含C型变形链球菌表面蛋白P1抗体的唾液膜对两种变形链球菌粘附的影响

用提纯的Ｓ．ｍｕｔａｎｓ ＭＴ６Ｒ细胞表面蛋白Ｐ１加弗氏佐剂通过局部唾液腺和背部皮下注射免疫ＢＡＬＢ／Ｃ鼠，得到含高效价Ｐ１抗体的大鼠唾液。用含Ｐ１抗体的唾液包被羟磷灰石微珠，观察含在唾液膜中的Ｃ型变链Ｐ１抗体对Ｓ．ｍｕｔａｎｓ ＭＴ６Ｒ和Ｓ．ｓｏｂｒｉｎｕｓ ６７１５在ＨＡ表面粘附的影响。     

变形链球菌葡糖基转移酶基因缺陷型突变株特征的研究

为了探讨各种葡糖基转移酶（ｇｌｕｃｏｓｙｌｔｒａｎｓｆｅｒａｓｅ，ＧＴａｓｅ）在细菌粘附中的作用，对变形链球菌ＭＴ８１４８ＧＴａｓｅ缺陷型突变株的特征进行了研究，明确了ＧＴａｓｅ的致龋作用。结果表明，与亲代菌相比，ＧＴａｓｅ缺陷株在蔗糖存在下对玻璃壁的粘附能力明显下降，产生的非水溶性葡聚糖明显少于亲代菌；合成非水溶性葡聚糖的ＧＴａｓｅ酶活力也明显低于ＭＴ８１４８。采用抗ＣＡＧＴａｓｅ多克隆抗体及抗ＧＴＦＩ或ＧＴＦＳＩ单克隆抗体的Ｗｅｓｔｅｒｎ印迹结果显示，各ＧＴａｓｅ缺陷株缺乏相应的ＧＴａｓｅ酶，同时也证实了ＣＡＧＴａｓｅ与ＣＦＧＴａｓｅ为二种相互类似但仍具一定特异性抗原决定簇的酶     

变形链球菌Ⅰ型和Ⅲ型合成葡聚糖的研究

目的 通过对Ⅰ型ＪＢＰ（ｃ型）和Ⅲ型６７１５（ｇ型）变链菌在不同状态葡糖基转移酶（ＧＴＦ）所合成的水不溶性和葡聚糖（ＩＧ）和水溶性葡聚糖（ＳＧ）的测定来研究两种变链菌的ＧＴＦ－Ⅰ和ＧＴＦ－Ｓ是否存在差异。方法 制备ＪＢＰ和６７１５的含酶上清液（ｃｅｌｌ－ｆｒｅｅ ｅｎｚｙｍｅ）及含酶提取液（ｃｅｌｌ－ｂｏｕｎｄ ｅｎｚｙｍｅ），以蔗糖为底物，恒温培养后分别收集ＩＧ和ＳＧ。     

Isolation of egg yolk IgY from hens immunized with S.m …

目的：从鸡蛋中提取特异性抗体,鉴定抗体性质并分析其蛋白质含量,以探索一种制备被动免疫防龋抗体的制备方法。方法变形链球菌ＧＴＦ高表达株Ｓ．ｍｕｔａｎｓＢ２９－３３及茸毛链球菌Ｓ．ｓｏｂｒｉｎｕｓ６７１５免疫母鸡,采用硫酸铵沉淀法提取鸡蛋黄抗体,ＳＤＳ－ＰＡＧＥ凝胶扫描鉴定抗体性质,分析其纯度并用分光光度法测蛋白质含量。结果：用硫酸铵沉淀法可提取出鸡蛋黄抗体,其性质为ＩｇＧ,蛋白质含量约为７．００ｍｇ     

变形链球菌毒力因子GTF多肽疫苗研究近况

变形链球菌是龋病主要的病原菌。变形链球菌定植至牙表面为产生龋齿关键的一步。这一过程由蔗糖依赖和蔗糖非依赖两种机制所介导 ,而后一机制与葡糖基转移酶(ＧＴＦ)催化蔗糖合成可溶性和不可溶性的葡聚糖有关[1] 。所以 ,ＧＴＦ被认为是变形链球菌最重要的两种毒力因子之一。它在致龋过程中起的重要作用使它成为发展防龋疫苗的研究对象[2 ] 。随着免疫学、分子生物学和基因工程技术的发展 ,对变链菌ＧＴＦ认识的逐步深入 ,利用ＧＴＦ分子中与某种特定功能有关并具有免疫原性的氨基酸序列制作的多肽疫苗 ,成为免疫防龋研究的热点。ＧＴＦ的…     

变形链球菌葡糖基转移酶缺陷突变株合成葡聚糖的扫描电镜观察

采用扫描电子显微镜观察在蔗糖存在下变链ＭＴ８１４８及其葡糖基转移酶缺陷突变株Ｂ２９、Ｂ５８和Ｂ３２合成葡聚糖的超微结构。结果在蔗糖存在下，变链ＭＴ８１４８及其ＧＴＦＳＩ缺陷株菌体表面和菌体间有大量丝状、不定形物质，而ＧＴＦＩ缺陷株Ｂ２９、ＧＴＦＩ和ＧＴＦＳＩ缺陷株Ｂ３２菌体表面光滑，菌体间无不定形物质，表明在变链ＭＴ８１４８、Ｂ５８菌体表面和菌体间的不定形物质为ＧＴＦ１酶产物－非水溶性葡聚糖。     

变形链球菌葡糖基转移酶缺陷突变株合成葡聚糖的扫描电…

采用扫描电子显微镜观察在蔗糖存在下变链ＭＴ８１４８及其葡糖基转移酶缺陷突变株Ｂ２９、Ｂ５８和Ｂ３２合成葡聚糖的超微结构。结果在蔗糖存在下，变链ＭＴ８１４８及其ＧＴＦＳＩ缺陷株菌体表面和菌体间有大量丝状、不定形物质，而ＧＴＦ１缺陷株Ｂ２９、ＧＴＦＩ和ＧＴＦＳＩ缺陷株Ｂ３２菌体表面光滑，菌体间无不定形物质，表明在变链ＭＴ８１４８、Ｂ５８菌体表面和菌体间的不定形物质为ＧＴＦＩ酶产物－非水溶性葡聚糖。     

变形链球菌传播和感染的研究进展

变形链球菌群细菌 (ｍｕｔａｎｓｓｔｒｅｐｔｏｃｏｃｃｉ,简称ＭＳ) ,特别是变形链球菌 (Ｓｔｒｅｐｔｏｃｏｃｃｕｓｍｕｔａｎｓ,简称变链菌 )和远缘链球菌 (Ｓ .ｓｏｂｒｉｎｕｓ)是人类最主要的致龋菌。预防ＭＳ的感染 ,干扰ＭＳ的传播途径 ,可有效减少这种微生物对人体的危害 ,因此 ,确定ＭＳ感染源以及探索其传播到易感者的方式 ,将有助于阻断或延迟ＭＳ的传播。近年来 ,随着以ＤＮＡ为基础的分子生物学方法的发展 ,ＭＳ在人类传播的研究进展方面迅速 ,本文就这方面的研究内容作一综述。1 ＭＳ的分子流行病学研究方法准确鉴定同种细…     

猛性龋儿童变链菌分离株的初始粘附能力

目的 :探讨猛性龋儿童变链菌和远缘链球菌临床分离株的初始粘附能力。方法 :采用唾液包被羟磷灰石 (SHA)及同位素标记方法 ,检测猛性龋、非猛性龋、无龋儿童变链菌 (各 6株 )和远缘链球菌 (猛性龋儿童6株 ,非猛性龋和无龋儿童各 3株 )临床株对SHA的粘附情况。结果 :各组变链菌分离株之间及各组远缘链球菌分离株之间对SHA的粘附率无显著差异 ;在无蔗糖条件下 ,各组远缘链球菌分离株对SHA的粘附百分率均低于各组变链菌 ,差异有显著性 (P <0 .0 5 )。结论 :变链菌的初始粘附能力强于远缘链球菌 ;猛性龋儿童变链菌和远缘链球菌临床株对SHA的初始粘附能力与非猛性龋及无龋儿童无差别。     

变形链球菌和远缘链球菌耐氟菌株粘附能力的体外研究

目的：探讨变形链球菌和远缘链球菌耐氟突变后,其粘附能力的改变。方法用同位素标记方法测定变形链球菌,远缘链球菌亲代及耐氟菌株粘附至唾液包被羟基磷灰石（ＳＨＡ）表面的粘附量及粘附百分率,并比较两者的粘附能力。结果变形链球菌、远缘链球菌亲人菌株与耐菌株间粘附百分率无明显差异（Ｐ〉０．０５）;远缘链球菌与变形链球菌相比,其粘附百分率偏低,差异有显著性（Ｐ〈０．０１）结论变形链球菌与远缘链球菌耐氟菌株粘附至     

In vitro inhibitory effects of Polygonum cuspidatum on bacterial viability and virulence factors of Streptococcus mutans and Streptococcus sobrinus

OBJECTIVES: Polygonum cuspidatum has been used in Korean folk medicine to improve oral hygiene. This study was performed to evaluate the effects of methanol extract from root of P. cuspidatum (MEP) on bacterial viability and the virulence factors of Streptococcus mutans and Streptococcus sobrinus. METHODS: To test the effects of MEP on bacterial viability, we determined the minimal inhibitory concentration (MIC) and minimal bactericidal concentration (MBC) against 20 bacterial strains, including S. mutans and S. sobrinus, using a micro-dilution assay. In case of S. mutans and S. sobrinus, the assays for time-kill and bacterial growth rate at sub-MIC concentrations were also performed. To determine effects of the extract on the virulence factors of S. mutans and S. sobrinus, the assays for sucrose-dependent adherence, water-insoluble glucan formation, glycolytic acid production, and acid tolerance were performed at sub-MIC levels. Phytochemical analysis for constituents of MEP was carried out. RESULTS: MEP showed a broad antibacterial range (MIC 0.5-4 mg/ml). The MBC was two to four times higher than the MIC. The time-kill curves showed S. mutans and S. sobrinus were significantly killed after 1h of incubation. At sub-MIC levels, doubling times of S. mutans and S. sobrinus dose-dependently increased up to 211% and 123%, respectively. At sub-MIC levels, MEP also showed inhibitory effects on the virulence factors of S. mutans and S. sobrinus in a dose-dependent fashion. Phytochemical analysis revealed the presence of alkaloids, sterol/terpenes, tannins, flavonoids, and carbohydrates. CONCLUSION: These data indicate that MEP has inhibitory effects on bacterial viability at higher concentrations (> or =MIC) and the virulence factors of S. mutans and S. sobrinus at sub-MIC concentrations, suggesting that it might be useful for the control of dental plaque formation and subsequent dental caries formation.     

Human strains of mutans streptococci show different cariogenic potential in the hamster model

The cariogenic potential of fresh humans strains of Streptococcus mutans and Streptococcus sobrinus was examined in hamsters maintained on a high sucrose diet. The strains 1B 16 (S. mutans) and B13 (S. sobrinus) isolated 20-25 yrs ago served as positive controls. Three series of experiments were run. Some strains were tested once and some strains were tested in all 3 experiments. The animals infected with S. sobrinus strains generally showed lower caries scores than those infected with S. mutans strains. Among the groups infected with the different strains of S. mutans the caries scores varied. In one of the experiments the caries score of the animals infected with one of the Icelandic S. mutans strains was significantly higher than that of the positive control group and the group infected with one of the fresh Swedish isolates. Thus, different strains of mutans streptococci where shown to induce varying caries activity in the hamster model.     

Binding of streptococci of the "mutans" group to type 1 collagen associated with apatitic surfaces

The present study surveyed the ability of 21 strains of cariogenic streptococci of the "mutans" group to bind to human type 1 collagen adsorbed on hydroxyapatite (HA) surfaces. All strains of Streptococcus cricetus (serotype a) and Streptococcus rattus (serotype b) tested attached strongly to collagen-treated HA (C-HA). Streptococcus mutans strains of serotype c and Streptococcus sobrinus strains of serotype d or g attached poorly; or not at all, to C-HA. S. mutans strains OMZ-175 (serotype f) and B14 (serotype e) were exceptions which exhibited significant binding to C-HA. Binding of S. cricetus AHT and S. rattus LB-1 to C-HA occurred in a dose-dependent manner, and was specific since it was inhibited by the presence of soluble collagen, but not by egg albumin, or by human fibrinogen or fibronectin. Binding occurred maximally between pH 6 and 8 and was not affected by any of several sugars tested. Trypsin treatment the streptococci had little affect, but heating at 100 degrees C reduced their ability to bind to C-HA.     

变形链球菌和远缘链球菌耐氟菌株DNA G+C含量测定

目的　探讨变形链球菌和远缘链球菌耐氟菌株基因型是否发生改变。方法　采用高效液相色谱法测定变形链球菌和远缘链球菌及相应耐氟菌株DNA碱基含量,即DNAG＋C摩尔百分比(mol%)。结果　四株耐氟菌株DNAG＋Cmol%为54.45,44.86,54.41,55.39;相应亲代菌株G+Cmol%为35.8,37.4,43.35和46.62。两者明显不同,差值大于5％。结论　变形链球菌和远缘链球菌的耐氟菌株已发生基因型改变,且可能包括多个基因的突变。     

绿茶多酚和氟化钠对变链菌形态学及酶学的影响

目的：为深入探讨绿茶多酚防龋机理。方法：以氟化钠为对照,观察了绿茶多酚对变链菌形态学及其蔗糖酶、葡糖基转移酶（ＧＴＦ）和乳酸脱氢酶（ＬＤＨ）的影响,同时检测了培养液中蛋白含量和ｐＨ值的改变。结果：绿茶多酚和氟化钠对ＧＴＦ均有抑制作用,前者强于后者,而对蔗糖酶影响不明显。绿茶多酚对ＬＤＨ无明显影响,不能阻止培养基ｐＨ的下降,对菌液蛋白总量无明显改变;而氟化钠对ＬＤＨ有影响,明显减少培养基ｐＨ值的下降,且使菌液蛋白总量明显增加。结论：绿茶多酚对ＧＴＦ具有很强的抑制作用,这是其抗变链菌致病作用的主要途径之一。     

小沟聚合物探针实时检测变形链球菌和远缘链球菌

目的　寻求一种快速检测变形链球菌(S.mutans)和远缘链球菌(S.sobrinus)的方法,研究变形链球菌群在儿童猛性龋患者口中的分布。方法　设计并合成针对S.mutans和S.sobrinus葡糖基转移酶基因(gtf)的特异性引物和小沟聚合物探针(MGB探针),对9株变形链球菌群参考菌株直接提取DNA及扩增纯培养后提取DNA分别进行检测,比较二者检测结果的异同。采集92例猛性龋儿童菌斑样本,用MGB探针进行实时检测。结果　采用MGB 探针可以特异性地鉴别S.mutans和S.sobrinus,直接检测和扩增纯培养后的定性检测结果完全一致,前者荧光出现的时间略迟。92例猛性龋患儿中S.mutans检出率为96.7%,S.sobrinus检出率为32.6%,所有检出S.sobrinus的菌斑样本均可检出S.mutans。结论　采用特异性MGB探针方法可以对菌斑中S.mutans和S.sobrinus进行实时检测, 提高检测效率。     

微滴数字聚合酶链反应检测婴儿唾液中变形链球菌和远缘链球菌的研究

目的 使用微滴数字PCR检测婴儿口腔内变形链球菌和远缘链球菌,评价其可行性.方法 以变形链球菌c型和远缘链球菌ATCC 27607标准菌株基因设计引物,对123名5~8个月婴儿唾液样本分别进行微滴数字PCR、巢氏PCR和常规PCR反应,检测婴儿唾液中变形链球菌和远缘链球菌.结果 微滴数字PCR定性、定量检测变形链球菌和远缘链球菌标准菌,1×10-4 ng/μl靶细菌的拷贝数达到4.8×104和3.6×104,灵敏度可达0.01%;巢氏PCR检测灵敏度为0.01%;常规PCR检测灵敏度为0.1%.用微滴数字PCR、巢氏PCR和常规PCR检测婴儿唾液中变形链球菌检出率分别为78.9%、33.3%和6.5%;远缘链球菌检出率分别为11.4%、4.1%和0,微滴数字PCR检出率明显高于常规PCR和巢氏PCR(P<0.05).结论 微滴数字PCR能早期检测婴儿口腔中变形链球菌和远缘链球菌,该方法具有较高的灵敏性和特异性,有广泛的临床应用前景.