盐酸胍法构建骨肉瘤细胞适配体ssDNA文库可行性
Feasibility of Guanidine Hydrochloride Method for ssDNA Library Construction of Osteosarcoma Aptamer
魏唐文 1魏兵 1黄丹菊 2梁昭珺 2张宇飞 2王秀娟 2傅桂莲 3梁骥2
作者信息
- 1. 桂林医学院医学检验学院,广西 桂林 541004;桂林医学院公共卫生学院,广西 桂林 541004
- 2. 桂林医学院医学检验学院,广西 桂林 541004
- 3. 北华大学医学技术检验学院,吉林 吉林 132013
- 折叠
摘要
目的 探讨链霉亲和素-生物素(Streptavidin-Biotin,STV-Biotin)系统偶联盐酸胍(Guanidine hydrochloride,G-HCl)法构建骨肉瘤细胞适配体文库的可行性.方法 将备选寡核苷酸文库与目标细胞(骨肉瘤细胞U2-OS)进行选择性结合,应用PCR法将与细胞结合的寡核苷酸扩增成双链DNA(Double stranded DNA,dsDNA);dsDNA被生物素标记后与 Streptavidin Sepharose(S.S)亲和层析介质结合,应用G-HCl法解离并纯化,制备目标单链寡核苷酸(Single stranded DNA,ssDNA)文库,以经典NaOH法为对照;将两种方法制备的单链文库分别与U2-OS结合,进行亲和性、回收率比较.结果 G-HCl方法获得的筛选文库ssDNA与U2-OS亲和性及ssDNA回收率高于经典NaOH法.结论 STV-Biotin系统偶联G-HCl成功制备了用于骨肉瘤细胞的适配体,为肿瘤适配体ssDNA文库构建提供了新的思路与方法.
Abstract
Objective Exploring feasibility of Streptavidin-Biotin(STV-Biotin)system coupled with Guanidine hydrochloride(G-HCl)method for construction of an osteosarcoma cell aptamer ssDNA library.Methods The candidate oligonucleotide library ssDNA was selectively bound to target cell(osteosarcoma cell U2-OS),and the oligonucleotides were amplified into double-stranded DNA(dsDNA)by PCR,dsDNA were labeled with biotin and combined with Streptavidin Sepharose(S.S)affinity chromatography.The target single-stranded DNA(ssDNA)library was prepared by G-HCl method and purified.The classical NaOH method was used for control.The single strand libraries prepared by the two methods were compared with osteosarcoma cells for compatibility and recovery.Results Screening library ssDNA obtained by G-HCl method have higher affinity to U2-OS and higher recovery of ssDNA than classical NaOH method.Conclusion STV-Biotin system coupled with G-HCl is a successful attempt for the preparation of osteosarcoma cell aptamer,which provides a new idea and method for the const-ruction of tumor aptamer ssDNA library.
关键词
适配体/骨肉瘤细胞/盐酸胍/PCR/单链寡核苷酸Key words
aptamer/osteosarcoma cells/Guanidine hydrochloride/PCR/ssDNA引用本文复制引用
出版年
2025
国家科技期刊平台
NSTL
万方数据