H22肿瘤细胞aptamer的筛选与结构分析

Screening,cloning and structure analysis of aptamer binding to H22 tumor cells

目的:获得与H22肿瘤细胞特异性结合的aptamer。方法:利用SELEX技术,以小鼠DC细胞为反筛选细胞,以H22肿瘤细胞为靶细胞,从体外合成的80bp随机单链DNA文库中筛选出能与H 22肿瘤细胞特异结合的aptamer。采用荧光标记引物法检测aptamer与H 22肿瘤细胞的亲和力。所获得的aptamer采用MACAWv2.05软件进行aptamer序列的一级结构同源序列比较,用DNASIS v2.5软件计算分析序列最低二级结构能量值,获得其二级结构模拟图。结果:本实验设计的文库序列:5′-CGTCGCTGCACATTCCG-N46-CGCACAGCTGGGA GTAC-3′具有较高的扩增效率,适合于aptamer与以细胞为靶物质的筛选。经过11轮循环筛选,随机单链DNA文库与靶细胞结合的荧光强度从1%上升到59%,结合曲线进入平台期,表明结合已基本处于稳定状态,因此可以判断aptamer与靶细胞的结合基本已经处于饱和状态。对所获得的32个aptam er进行测序,然后进行一级结构和二级结构分析。一级结构分析获得5个保守序列:AGGGA、AGAAGG、GTGAXAA、ATAGT、CAAGG,其余10个aptamer无同源序列。二级结构分析表明,aptamer形成的茎环、凸环结构可能是与H 22肿瘤细胞特异性结合的结构基础。亲和力检测结果表明第24号aptamer具有最强的亲和力。结论:利用随机单链寡核苷酸文库成功获得与H22肿瘤细胞特异结合的aptamer,其一级和二级结构与亲和力密切相关。

Objective：To obtain the single-stranded DNA aptamer that specially,＇ binds H22 tumor cells.Methods：H22 tumor cells were acted as target and rat DC cells were acted as counter-target by using the process of systematic evolution of ligands by exponential enrichment,which was abbreviated as SELEX.Aptamer that specially bind H22 cancer cells were selected in vitro from 80bp random single-stranded DNA library. Aptamer affinity with H22 cancer cell was detected by the fluorescence-labeled primers method. The acquired aptamer was analyzed its primary structure isogenetic sequence comparisonby MACAW v2.05 software, and was predicted its secondary structure by using DNASIS v2.5 Software.Results：Library for this experiment 5＇ - CGTCGCTGCACATTCCG - （ 46N ）- CG- CACAGCTGGGAGTAC -5＇ had a higher PCR amplification efficacy, was fit for the selection which act cell as target. After 11 cycles selection, fluorescence intensity of random single-stranded DNA binding with target cells was changed from 1% to 59%, binding curve entered platform stage, i.e., binding level was steady state, the binding that aptamer with target cells was in saturation state. There were 5 conserved sequenced and the structure analysis revealed that the stem-loops or bulge was the main motif in the interaction between aptamers and H22 cancer cell.Conclusion：Aptamers against H22 cancer cell have been identified by SELEX methods from an 80 bp single-stranded DNA random library.