留言板

尊敬的读者、作者、审稿人, 关于本刊的投稿、审稿、编辑和出版的任何问题, 您可以本页添加留言。我们将尽快给您答复。谢谢您的支持!

姓名
邮箱
手机号码
标题
留言内容
验证码
x

CA125抗原核酸适体的筛选及鉴定

黄百海 何雷 李保林 修尘林 赵运旺

黄百海, 何雷, 李保林, 修尘林, 赵运旺. CA125抗原核酸适体的筛选及鉴定[J]. 分子影像学杂志, 2019, 42(1): 52-56. doi: 10.12122/j.issn.1674-4500.2019.01.13
引用本文: 黄百海, 何雷, 李保林, 修尘林, 赵运旺. CA125抗原核酸适体的筛选及鉴定[J]. 分子影像学杂志, 2019, 42(1): 52-56. doi: 10.12122/j.issn.1674-4500.2019.01.13
Baihai HUANG, Lei HE, Baolin LI, Chenlin XIU, Yunwang ZHAO. Screening and identification of aptamers to CA125 antigen[J]. Journal of Molecular Imaging, 2019, 42(1): 52-56. doi: 10.12122/j.issn.1674-4500.2019.01.13
Citation: Baihai HUANG, Lei HE, Baolin LI, Chenlin XIU, Yunwang ZHAO. Screening and identification of aptamers to CA125 antigen[J]. Journal of Molecular Imaging, 2019, 42(1): 52-56. doi: 10.12122/j.issn.1674-4500.2019.01.13

CA125抗原核酸适体的筛选及鉴定

doi: 10.12122/j.issn.1674-4500.2019.01.13
基金项目: 国家自然科学基金(30960104;30770639)
详细信息
    作者简介:

    黄百海,本科,E-mail: 2840252124@qq.com

    通讯作者:

    赵运旺,硕士,助理研究员,E-mail: zhaoyunwang1234@163.com

Screening and identification of aptamers to CA125 antigen

  • 摘要: 目的 建立一种基于核酸适体快速检测肿瘤血清标志物CA125抗原的方法。 方法 以CA125抗原为筛选靶标,CA199抗原为反筛选靶标,羧基化琼脂磁珠为筛选介质,利用指数级富集配体的系统进化技术筛选其高特异性核酸适体,利用生物信息学方法对核酸适体进行序列分析和二级结构预测,荧光定量法分析核酸适体的亲和力,ELONA法测定核酸适体的特异性。 结果 经过6轮消减SELEX筛选,次级ssDNA文库的亲和力逐渐提高,特异性次级ssDNA文库逐轮富集,经二代测序,获得3条拷贝数>1000的核酸序列(Apt-1、Apt-2、Apt-3),可特异性识别CA125抗原,测定Kd值,分别为41.02±4.9、19.06±9.0、10.02±5.0 nmol/L,Apt-3亲和力最高。 结论 功能性核酸适体Apt-3能高特异性识别CA125抗原,为肿瘤血清标志物CA125检测提供了一种新的、快速、有效的工具和手段。

     

  • 图  1  1-6轮ssDNA文库亲和力测定结果

    图  2  1-6轮ssDNA PCR产物琼脂糖凝胶电泳检测结果

    图  3  核酸适体同源性分析结果

    图  4  核酸适体二级结构预测结果

    图  5  核酸适体亲和力检测结果

    图  6  3条核酸适体特异性分析结果

    表  1  核酸适体测序结果

    核酸适体 碱基序列 Kd(nmol/L) ΔG(kcal·mol–1
    Apt-1 5’- CTATAGCAATGGTACGGTACTTCCCAATGGTACGGTACGGCCAA
    GCTAACCCTCATGTGCGCGC CAAAAGTGCACGCTACTTTGCTAA-3’
    41.02±4.9 –16.6
    Apt-2 5’- CTATAGCAATGGTACGGTACTTCCTACGACTCACAATAGGAGCT
    AACCCTCATCTGCGCGCTCC CAAAAGTGCACGCTACTTTGCTAA-3’
    19.06±9.0 –10.2
    Apt-3 5’- CTATAGCAATGGTACGGTACTTCCAAGCTGACCCTTACCTGCGC
    GCTCCACCCTCATCTACGCG CAAAAGTGCACGCTACTTTGCTAA-3’
    10.02±5.0 –15.2
    下载: 导出CSV
  • [1] Bast RC, Klug TL, St John E, et al. A radioimmunoassay using a monoclonal antibody to monitor the course of epithelial ovarian cancer[J]. N Engl J Med, 1983, 309(15): 883-7 doi: 10.1056/NEJM198310133091503
    [2] Huang Z, Zhai XM, Wang H, et al. Simultaneous quantitation of carbohydrate antigen 125 and carcinoembryonic antigen in human serum via time-resolved fluoroimmunoassay[J]. Clinica Chimica Acta, 2018, 483(10): 222-6
    [3] Kaaks R, Fortner RT, Huesing AA, et al. Tumor-associated autoantibodies as early detection markers for ovarian cancer? A prospective evaluation[J]. Int J Cancer, 2018, 143(3): 515-26 doi: 10.1002/ijc.v143.3
    [4] Liang C, Shi S, Meng Q, et al. MiR-29a,targeting caveolin 2 expression,is responsible for limitation of pancreatic cancer metastasis in patients with normal level of serum CA125[J]. Int J Cancer, 2018, 12(4): 482-9
    [5] Nishimoto-Kakiuchi A, Netsu S, Okabayashi S, et al. Spontaneous endometriosis in cynomolgus monkeys as a clinically relevant experimental model[J]. Hum Reprod, 2018, 33(7): 1228-36 doi: 10.1093/humrep/dey095
    [6] Zhou H, Dong A, Xia H, et al. Associations between CA19-9 and CA125 levels and human epidermal growth factor receptor 2 overexpression in patients with gastric cancer[J]. Oncol Lett, 2018, 16(1): 1079-86
    [7] Hamd-Ghadareh S, Salimi A, Parsa S, et al. Simultaneous biosensing of CA125 and CA15-3 tumor markers and imaging of OVCAR-3 and MCF-7 cells lines via bi-color FRET phenomenon using dual blue-green luminescent Carbon dots with single excitation wavelength[J]. Int J Biol Macromol, 2018, 118(A): 617-28
    [8] Zhang F, Wang JJ, Zheng XY, et al. Clinical value of jointly detection pleural fluid Midkine, pleural fluid adenosine deaminase, and pleural fluid carbohydrate antigen 125 in the identification of nonsmall cell lung cancer-associated malignant pleural effusion[J]. J Clin Lab Anal, 2018, 32(8): e22576-83 doi: 10.1002/jcla.2018.32.issue-8
    [9] Mckeague M, Mcconnell EM, Cruz-Toledo JA, et al. Analysis of in vitro aptamer selection parameters[J]. J Mol Evol, 2015, 81(5/6, SI): 150-61
    [10] Tuerk C, Gold L. Systematic evolution of ligands by exponential enrichment: RNA ligands to bacteriophage T4 DNA polymerase[J]. Science, 1990, 249(4968): 505-10 doi: 10.1126/science.2200121
    [11] Wheeler LA, Trifonova R, Vrbanac VA, et al. Inhibition of HIV transmission in human cervicovaginal explants and humanized mice using CD4 aptamer-siRNA chimeras[J]. J Clin Invest, 2011, 121(6): 2401-12 doi: 10.1172/JCI45876
    [12] Fang XH, Sen A, Vicens M, et al. Synthetic DNA aptamers to detect protein molecular variants in a high-throughput fluorescence quenching assay[J]. Chembiochem, 2003, 4(9): 829-34 doi: 10.1002/cbic.v4:9
    [13] Cai D, Xu D, Zhang Q, et al. Classification of lung cancer using ensemble-based feature selection and machine learning methods[J]. Molecular biolystems, 2014, 464(7291): 1071-6
    [14] Gilboa E, McNamara J, Pastor F. Use of oligonucleotide aptamer ligands to modulate the function of immune receptors[J]. J Ame Assoc Cancer Res, 2013, 43(19): 1054-62
    [15] Hasegawa H, Sode K, Ikebukuro K. Selection of DNA aptamers against VEGF(165) using a protein competitor and the aptamer blotting method[J]. Biotechnol Lett, 2008, 30(5): 829-34 doi: 10.1007/s10529-007-9629-6
    [16] Ireson CR, Kelland LR. Discovery and development of anticancer aptamers[J]. Mol Cancer Ther, 2006, 5(12): 2957-62 doi: 10.1158/1535-7163.MCT-06-0172
    [17] Jun YP, Ye LC, Ju RC, et al. Gemcitabine-Incorporated G-QuadruplexAptamer for targeted drug delivery into pancreas cancer[J]. Mol Ther Nucleic Acids, 2018, 12(8): 543-53
    [18] Visintin I, Feng Z, Longton G, et al. Diagnostic markers for early detection of ovarian cancer[J]. Clin Cancer Res, 2008, 14(4): 1065-72 doi: 10.1158/1078-0432.CCR-07-1569
    [19] Yang JM, Dou BT, Yuan R, et al. Aptamer/protein proximity Binding-Triggered molecular machine for amplified electrochemical sensing of thrombin[J]. Anal Chem, 2017, 89(9): 5138-43 doi: 10.1021/acs.analchem.7b00827
    [20] Li H, Arroyo-Curras N, Kang D, et al. Dual-Reporter drift correction to enhance the performance of electrochemical Aptamer-Based sensors in whole blood[J]. J Am Chem Soc, 2016, 138(49): 15809-12 doi: 10.1021/jacs.6b08671
    [21] 葸玉琴, 赵运旺, 李东东, 等. 胃癌血清适配体的筛选及其鉴定[J]. 南京医科大学学报:自然科学版, 2016, 36(11): 1306-12
    [22] Wu YX, Kwon YJ. Aptamers: the " evolution”of SELEX[J]. Methods, 2016, 106(15): 21-8
    [23] Mckeague M, De Girolamo A, Valenzano SA, et al. Comprehensive analytical comparison of strategies used for small molecule aptamer evaluation[J]. Anal Chem, 2015, 87(17): 8608-12 doi: 10.1021/acs.analchem.5b02102
  • 加载中
图(6) / 表(1)
计量
  • 文章访问数:  626
  • HTML全文浏览量:  419
  • PDF下载量:  9
  • 被引次数: 0
出版历程
  • 收稿日期:  2018-09-04
  • 刊出日期:  2019-01-01

目录

    /

    返回文章
    返回

    关于《分子影像学杂志》变更刊期通知

    各位专家、作者、读者:

    为了缩短出版时滞,促进科研成果的快速传播,我刊自2024年1月起,刊期由双月刊变更为月刊。本刊主要栏目有:基础研究、临床研究、技术方法、综述等。

    感谢各位专家、作者、读者长期以来对我刊的支持与厚爱!

    南方医科大学学报编辑部

    《分子影像学杂志》

    2023年12月27日