南方医科大学学报 ›› 2013, Vol. 33 ›› Issue (05): 738-.

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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

王成龙,胡丹阳,刘佼佼,李少华,苏东华,席庆,储冰峰,夏伟,赵强,丁红梅,罗燕萍,杨继勇,邓斌,徐娟,邵宁生   

  • 出版日期:2013-05-20 发布日期:2013-05-20

Selection and identification of ssDNA aptamers specific to clinical isolates of Streptococcus mutans strains with different cariogenicity

  • Online:2013-05-20 Published:2013-05-20

摘要: 目的筛选不同致龋性变形链球菌临床分离株差异ssDNA配基并对其进行鉴定。方法利用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,通过放射性同位素、流式细胞术、基因克隆测序、MEME
在线软件和RNA structue 分析软件分析配基的一、二级结构并对筛选得到的配基进行鉴定。结果经过9轮消减SELEX筛选,
放射性同位素检测文库已富集;流式细胞术检测经过测序和分析得到12条配基中的H1、H16、H4、L1、L10和H19与高致龋变形
链球菌临床分离株特异结合,不与低致龋变形链球菌临床分离株特异结合,其中,H19 的特异性结合程度最强,解离常数为
69.45±38.53 nmol/L。结论筛选获得特异识别高致龋变形链球菌临床分离株的ssDNA配基。

Abstract: Objective To select and identify ssDNA aptamers specific to Streptococcus mutans strains with different cariogenicity
isolated from clinical specimens. Methods Subtractive SELEX technology targeting the whole intact cells was used to screen
for ssDNA aptamers specific to the clinical isolates Streptococcus mutans strains with different cariogenicity. Radioactive
isotope, flow cytometry, gene cloning and sequencing, MEME online software and RNA structure analysis software were
employed to analyze the first and secondary structures of the aptamers and identify the screened aptapers. Results Detection
by radioactive isotope showed sufficient pool enrichment after 9 rounds of subtractive SELEX. Flow cytometry showed that the
selected aptamers H1, H16, H4, L1, L10 and H19 were capable of binding specifically with highly cariogenic Streptococcus
mutans strains but not with strains with a low cariogenicity. The aptamer H19 had the strongest binding capacity to highly
cariogenic Streptococcus mutans strains, with a dissociation constant of 69.45±38.53 nmol/L. Conclusion We have obtained the
ssDNA aptamers specific to the clinical isolates of highly cariogenic Streptococcus mutans strains.