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作 者:魏唐文 魏兵 黄丹菊 梁昭珺 张宇飞 王秀娟 傅桂莲[3] 梁骥 WEI Tangwen;WEI Bing;HUANG Danju;LIANG Zhaojun;ZHANG Yufei;WANG Xiujuan;FU Guilian;LIANG Ji(School of Medical Laboratory,Guilin Medical University,Guilin 541004,China;School of Public Health,Guilin Medical University,Guilin 541004,China;School of Medical Technology and Testing,Beihua University,Jilin 132013,China)
机构地区:[1]桂林医学院医学检验学院,广西桂林541004 [2]桂林医学院公共卫生学院,广西桂林541004 [3]北华大学医学技术检验学院,吉林吉林132013
出 处:《北华大学学报(自然科学版)》2025年第1期49-54,共6页Journal of Beihua University(Natural Science)
基 金:广西自然科学基金项目(2018GXSFAA138035);大学生创新创业训练计划项目(国家级,202310601054,校级,202210601237);广西高校中青年教师科研基础能力提升项目(2022KY0511);广西肿瘤免疫与微环境调控重点实验室开放课题(2202KF004)。
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。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.
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