【摘要】 目的建立一种通用的细胞-指数式富集的配体系统进化(Cell-SELEX)技术平台,用于筛选特异性结合于肿瘤细胞的DNA适配子。方法采用PCR法建立随机双链DNA(dsDNA)寡核苷酸文库,用卵白素包被的琼脂糖珠从dsDNA库分离单链DNA(ssDNA)文库;以体外培养的肿瘤细胞作为正筛选的靶标,以相同组织来源的正常对照细胞作为负筛选的靶标,消减法进行Cell-SELEX筛选和PCR扩增以获取DNA适配子;流式细胞仪监测适配子的富集效率(亲和力);采用常规分子生物学技术进行适配子的克隆、测序和序列分析。结果成功建立了ssDNA库,经过12轮Cell-SELEX筛选,获得了特异性结合于肿瘤细胞的DNA适配子;挑选120个阳性克隆送测序,鉴定出21个适配子,采用NCBI网站提供的Blastn程序进行精确比对未发现相似序列。结论本实验建立的技术平台可在较短时间内获取特异性结合于特定肿瘤细胞的DNA适配子。更多还原

【Abstract】 Objective To establish commonly used technical platform of cell-systematic evolution of ligands by exponential enrichment( cell-SELEX) for screening DNA aptamers specifically binding to tumor cells. Methods A random doublestranded DNA( dsDNA) pool was established by PCR, and single-stranded DNA( ssDNA) library was then separated from dsDNA pool by using avidin coated Sepharose. In order to get DNA aptamers for cancer cells, cell-SELEX and PCR were performed by using cultured gastric cancer cell line as positive screening target while normal gastric mucosal cell line as negative. Flow cytometry( FCM) was used to monitor the enrichment efficiency and affinity of aptamers. Cloning, sequencing, and sequence analysis of aptamers by conventional molecular biology techniques were also carried out. Results A random ssDNA pool was successfully established and specific aptamers of tumor cells were got after 12 runs’ cell-SELEX. Of 120 positive sequenced clones, 21 aptamers were identified. Accurate alignment of Blastn implied that not any similar sequence to 21 submitted aptamers was found in NCBI data base. Conclusion The article provided a technical platform for obtaining DNA aptamers specifically binding to tumor cells in a relatively short period of time.更多还原