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人骨肉瘤细胞适配体的早期筛选
引用本文:王华,梁骥,高远,孙娜,马艳,高丽君,夏薇.人骨肉瘤细胞适配体的早期筛选[J].北华大学学报(自然科学版),2015,16(3):311-315.
作者姓名:王华  梁骥  高远  孙娜  马艳  高丽君  夏薇
作者单位:北华大学附属医院,吉林 吉林,132011;吉林大学第一临床学院,吉林 长春,130021;北华大学医学检验学院,吉林 吉林,132013
基金项目:吉林省教育厅科学技术研究项目
摘    要:目的体外随机合成单链寡核苷酸备选文库,以骨肉瘤U-2 OS完整细胞作为靶点,筛选出与其结合的高特异性、高亲和性单链DNA(ss DNA).方法将体外合成随机碱基序列的单链DNA文库与人骨肉瘤U-2 OS细胞相结合,收获与靶细胞结合的单链DNA,通过优化PCR条件进行大量扩增后,应用生物素-链霉亲和素亲和层析柱分离出正链DNA.结果以10,14以及18个循环时扩增ds DNA产物最理想;应用生物素-链霉亲和素磁珠分离法制备的正链ss DNA纯度高,可有效保证次级文库的筛选.结论应用Cell-SELEX技术可成功筛选早期ss DNA文库,为后续获得针对人骨肉瘤U-2 OS细胞的高特异性适配体奠定实验基础.

关 键 词:人骨肉瘤  Cell-SELEX技术  适配体

Early Screening of Human Osteosarcoma Cell Aptamer
Wang Hua,Liang Ji,Gao Yuan,Sun Na,Ma Yan,Gao Lijun,Xia Wei.Early Screening of Human Osteosarcoma Cell Aptamer[J].Journal of Beihua University(Natural Science),2015,16(3):311-315.
Authors:Wang Hua  Liang Ji  Gao Yuan  Sun Na  Ma Yan  Gao Lijun  Xia Wei
Abstract:Objective Prepare single-stranded oligonucleotide ( ssDNA) with high affinity and specificity for Osteosarcoma U-2 OS,a target cell,by exponential enrichment strategy. Method The designed single-stranded DNA library were synthesized in vitro. After incubation of DNA library pool with U-2 OS cells, the ssDNAs binding to the target cells U-2 OS were amplified by PCR that optimum cycle had been determined. After amplification, separation ssDNA from dsDNA was finished by affinity chromatography with streptavidin-coated Sepharose beads. Results The products of dsDNA from 10,14 and 18 cycle showed desirable image. The single-stranded DNA ( ssDNA) separated from dsDNA by Streptavidin-coated Sepharose beads was of high purity,which was next round selection. Conclusion We have successfully selected the initial ssDNA pool by Cell-SELEX, which will be used for the development of DNA aptamer with high affinity for U-2 OS Osteosarcoma cell.
Keywords:human osteosarcoma  Cell-SELEX  aptamer
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