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

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

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。

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 nmolFh. Conclusion We have obtained the ssDNA aptamers specific to the clinical isolates of highly cariogenic Streptococcus mutans strains.