不同致龋性变形链球菌临床分离株差异ssDNA配基的筛选和鉴定
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摘要
龋病是最常见的人类口腔疾病,变形链球菌是引起人类龋病最主要的致病菌。变形链球菌致龋是一个多因素综合作用的结果,既包括已知的各种致龋因子,也可能还有目前仍不了解的其它因子或各种因子间的相互作用。因此,为了揭示变形链球菌致龋的机理,从细菌的整体水平研究变形链球菌是十分必要的,而SELEX技术为这一研究设想提供了实用工具。
指数式富集的配体系统进化技术(SELEX)是近年发展起来的研究核酸结构、功能及进化的一种组合化学技术,在基础医学、临床诊断和疾病治疗中都显现了广阔的应用前景。尤其是以完整细胞为靶子的消减SELEX技术的建立,使得研究不同细胞靶子上存在的未知差异靶分子成为可能。
本研究的目的是利用以完整细胞为靶子的消减SELEX技术筛选不同致龋性变形链球菌临床分离株差异ssDNA配基。
为了达到研究目的,我们首先利用细菌形态学、生化反应、自动微生物分析系统及分子生物学方法从11名受试者口腔牙菌斑中鉴定获得了性质确实可靠的5株变形链球菌临床分离株,其中3株来源于高龋患者,2株来源于无龋健康人；然后通过经典的体外致龋实验,即唾液包被羟基磷灰石粘附实验、合成胞外多糖实验、产酸及耐酸实验对筛选获得的变形链球菌临床分离株的致龋特性进行检测。综合考虑各实验结果并辅以统计学分析后,确定了临床分离株5为高致龋性变形链球菌,临床分离株4为低致龋性变形链球菌；采用全长为88nt、随机区由45个碱基构成的ssDNA文库,以变形链球菌临床分离株5为筛选靶子,变形链球菌临床分离株4为消减靶子,进行了9轮以完整细胞为靶子的消减SELEX筛选,获得了不同致龋性变形链球菌临床分离株差异ssDNA配基H19。H19与筛选靶子-变形链球菌临床分离株5的亲和力较高,解离常数为69.45±38.53nM。
本课题研究中筛选获得的不同致龋性变形链球菌临床分离株差异ssDNA配基有可能用于临床龋病易感性的预测,并且有可能发展成为抑制或阻断变形链球菌致龋的防龋制剂,为龋病防治提供新思路。
Streptococcus mutans is generally considered to be the principal aetiological agent for dental caries, which is the most prevalent bacterial infectious disease in oral cavity. Varieties of risk factors interact in this disease, not only including the cariogenic agents that have been known, but also including the unknown ones. In this regard, it is necessary to study on Streptococcus mutans as a whole to reveal the cariogenic mechanism of it. And SELEX can be a useful tool for this purpose.
SELEX (Systematic Evolution of Ligands by Exponential Enrichment) is a combinatorial chemistry technology for studying structure, function and molecular evolution of nucleic acid. The aptamers evolved from this in vitro selection process have a promising application in basic research, chlinical diagnosis and therapy. The recently establishment of subtractive SELEX method, which targets the whole intact cells, can even generate aptamers that differentiate target cells from counter-selected partners without the prior knowledge of the molecular differences.
The objective of this research is to select and identify different ssDNA aptamers specific to Streptococcus mutans isolated from caries-free and caries-active individuals by the subtractive SELEX.
Before the selection, we identified and obtained 5 clinical isolates of Streptococcus mutans from dental plaques of 11 subjects by morphology, biochemical events, automated microbiology system, and molecular biological methods. Among the isolates, there were 3 ones from caries-active individuals, while 2 from caries-free individuals. Then, we detected the cariogenic virulence characteristics of the isolates above by four classical in vitro cariogenic experiments, which were adherence analysis, extracellular polysaccharide synthesis assay, aciduricity and acidogenicity assays. Combination with statistics analysis, the results revealed that No.5 isolate was the high virulent strain, while No.4 isolate was the low one.
The subtractive SELEX selection against No.5 Streptococcus mutans isolate with an 88 nucleotides ssDNA library containing 45 random nucleotides flanked by invariant primer was done, followed by counter selection to No.4 Streptococcus mutans isolate. After 9 rounds selection, that targeted the whole intact cells, one aptamer named H19, which showed high specificity and affinity to No.5 Streptococcus mutans isolate, was identified with a Kd of 69.45±38.53nM.
Overall, it was possible that the ssDNA aptamers specific to Streptococcus mutans isolated from caries-free and caries-active individuals of our work might be used for clinical prediction to the susceptibility of dental caries, and might be a new idea for the prevention of dental caries.
指数式富集的配体系统进化技术(SELEX)是近年发展起来的研究核酸结构、功能及进化的一种组合化学技术,在基础医学、临床诊断和疾病治疗中都显现了广阔的应用前景。尤其是以完整细胞为靶子的消减SELEX技术的建立,使得研究不同细胞靶子上存在的未知差异靶分子成为可能。
本研究的目的是利用以完整细胞为靶子的消减SELEX技术筛选不同致龋性变形链球菌临床分离株差异ssDNA配基。
为了达到研究目的,我们首先利用细菌形态学、生化反应、自动微生物分析系统及分子生物学方法从11名受试者口腔牙菌斑中鉴定获得了性质确实可靠的5株变形链球菌临床分离株,其中3株来源于高龋患者,2株来源于无龋健康人；然后通过经典的体外致龋实验,即唾液包被羟基磷灰石粘附实验、合成胞外多糖实验、产酸及耐酸实验对筛选获得的变形链球菌临床分离株的致龋特性进行检测。综合考虑各实验结果并辅以统计学分析后,确定了临床分离株5为高致龋性变形链球菌,临床分离株4为低致龋性变形链球菌；采用全长为88nt、随机区由45个碱基构成的ssDNA文库,以变形链球菌临床分离株5为筛选靶子,变形链球菌临床分离株4为消减靶子,进行了9轮以完整细胞为靶子的消减SELEX筛选,获得了不同致龋性变形链球菌临床分离株差异ssDNA配基H19。H19与筛选靶子-变形链球菌临床分离株5的亲和力较高,解离常数为69.45±38.53nM。
本课题研究中筛选获得的不同致龋性变形链球菌临床分离株差异ssDNA配基有可能用于临床龋病易感性的预测,并且有可能发展成为抑制或阻断变形链球菌致龋的防龋制剂,为龋病防治提供新思路。
Streptococcus mutans is generally considered to be the principal aetiological agent for dental caries, which is the most prevalent bacterial infectious disease in oral cavity. Varieties of risk factors interact in this disease, not only including the cariogenic agents that have been known, but also including the unknown ones. In this regard, it is necessary to study on Streptococcus mutans as a whole to reveal the cariogenic mechanism of it. And SELEX can be a useful tool for this purpose.
SELEX (Systematic Evolution of Ligands by Exponential Enrichment) is a combinatorial chemistry technology for studying structure, function and molecular evolution of nucleic acid. The aptamers evolved from this in vitro selection process have a promising application in basic research, chlinical diagnosis and therapy. The recently establishment of subtractive SELEX method, which targets the whole intact cells, can even generate aptamers that differentiate target cells from counter-selected partners without the prior knowledge of the molecular differences.
The objective of this research is to select and identify different ssDNA aptamers specific to Streptococcus mutans isolated from caries-free and caries-active individuals by the subtractive SELEX.
Before the selection, we identified and obtained 5 clinical isolates of Streptococcus mutans from dental plaques of 11 subjects by morphology, biochemical events, automated microbiology system, and molecular biological methods. Among the isolates, there were 3 ones from caries-active individuals, while 2 from caries-free individuals. Then, we detected the cariogenic virulence characteristics of the isolates above by four classical in vitro cariogenic experiments, which were adherence analysis, extracellular polysaccharide synthesis assay, aciduricity and acidogenicity assays. Combination with statistics analysis, the results revealed that No.5 isolate was the high virulent strain, while No.4 isolate was the low one.
The subtractive SELEX selection against No.5 Streptococcus mutans isolate with an 88 nucleotides ssDNA library containing 45 random nucleotides flanked by invariant primer was done, followed by counter selection to No.4 Streptococcus mutans isolate. After 9 rounds selection, that targeted the whole intact cells, one aptamer named H19, which showed high specificity and affinity to No.5 Streptococcus mutans isolate, was identified with a Kd of 69.45±38.53nM.
Overall, it was possible that the ssDNA aptamers specific to Streptococcus mutans isolated from caries-free and caries-active individuals of our work might be used for clinical prediction to the susceptibility of dental caries, and might be a new idea for the prevention of dental caries.
引文
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