CE-SELEX法筛选Mucin16胞外域的核酸适配体

Screening and identification of an ss DNA aptamer targeting the extracellular domain of Mucin16 by CE-SELEX

目的 CA125与胃癌的腹腔转移及不良预后密切相关,文中旨在针对CA125所属糖蛋白Mucin16的胞外域,筛选针对其的单链DNA核酸适配体。方法利用毛细管电泳法筛选Mucin16胞外域-合成多肽的适配体,同时利用毛细管电泳定量测定每轮筛选的Kd值及所筛得的适配体与多肽的亲和力,激光共聚焦成像法对所筛得的适配体靶向癌细胞进行评价。结果经过5轮筛选,测序分析及亲和力测定,成功获得1条针对Mucin16胞外域多肽的最优单链DNA核酸适配体,Kd值达122.7 nm,并且在表达Mucin16的人卵巢癌细胞株的细胞膜上清晰可见绿色荧光信号,证实所筛适配体能够结合Mucin16的细胞膜胞外域。结论利用毛细管电泳法成功筛选出针对Mucin16胞外域的核酸适配体,在为胃癌腹腔转移的诊疗中提供了新的靶向分子。

Objective CA125 has been proved to be closely related to peritoneal metastasis of gastric cancer. This study aimed to screen and identify a novel ss DNA aptamer targeting the extracellular domain of Mucin16. Methods Using capillary electrophoresis,we screened the aptamers targeting the synthetic peptide of the extracellular domain of Mucin16 and quantitatively determined the Kd value of each cycle and the affinity of the aptamers for the synthetic peptide by CE-SELEX. Then we evaluated the ability of the obtained aptamers to target cancer cells using confocal laser imaging. Results After five cycles of screening,sequencing,affinity determination,we obtained an ss DNA aptamer targeting the extracellular domain of Mucin16,with a Kd value of 122. 7 nm and visible green fluorescent signals on the cell membrane of the human ovarian cancer cell line expressing Mucin16,but not on that of the normal hepatocytes not expressing Mucin16. This confirmed the binding ability of the aptamer to the extracellular domain of Mucin16.Conclusion A novel aptamer targeting the synthetic peptide of the extracellular domain of Mucin16 was successfully obtained by capillary electrophoresis,which could be used as a new agent in the diagnosis and treatment of peritoneal metastasis of gastric cancer.