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肺癌患者血清特异性核酸适配体的筛选及鉴定

邸娅 赵运旺 卢坤玲 何雷 徐欢 郑岳

邸娅, 赵运旺, 卢坤玲, 何雷, 徐欢, 郑岳. 肺癌患者血清特异性核酸适配体的筛选及鉴定[J]. 分子影像学杂志, 2018, 41(3): 334-340. doi: 10.3969/j.issn.1674-4500.2018.03.12
引用本文: 邸娅, 赵运旺, 卢坤玲, 何雷, 徐欢, 郑岳. 肺癌患者血清特异性核酸适配体的筛选及鉴定[J]. 分子影像学杂志, 2018, 41(3): 334-340. doi: 10.3969/j.issn.1674-4500.2018.03.12
Ya DI, Yunwang ZHAO, Kunling LU, Lei HE, Huan XU, Yue ZHENG. Sreening and identification of serum specific aptamers for lung cancer[J]. Journal of Molecular Imaging, 2018, 41(3): 334-340. doi: 10.3969/j.issn.1674-4500.2018.03.12
Citation: Ya DI, Yunwang ZHAO, Kunling LU, Lei HE, Huan XU, Yue ZHENG. Sreening and identification of serum specific aptamers for lung cancer[J]. Journal of Molecular Imaging, 2018, 41(3): 334-340. doi: 10.3969/j.issn.1674-4500.2018.03.12

肺癌患者血清特异性核酸适配体的筛选及鉴定

doi: 10.3969/j.issn.1674-4500.2018.03.12
详细信息
    作者简介:

    邸娅:邸 娅,硕士,E-mail: 47352569@qq.com

    通讯作者:

    郑 岳,主任医师,教授,E-mail: zhengyueqhd@sina.com

Sreening and identification of serum specific aptamers for lung cancer

  • 摘要: 目的 以磁珠为筛选介质,利用实时定量-PCR和消减SELEX技术从肺癌血清中筛选得到高特异性、强亲和力的肺癌血清核酸适配体。 方法 以磁珠为载体、肺癌病人血清为筛选的靶标分子,通过消减SELEX技术及实时定量PCR技术,从随机ssDNA文库中筛选出与肺癌血清特异性结合的aptamers,将得到的第9轮富集文库扩增为dsDNA,送上海生工直接对筛选得到的核酸适配体库进行高通量测序。 结果 经过9轮筛选得到4条与肺癌血清高特异性结合的aptamers,测序结果显示均为不同序列。 结论 验证筛选出的与肺癌血清结合的aptamer,特异性检测表明,筛选得到的肺癌血清核酸适配体与肺癌血清的结合解离常数均在纳摩尔级水平,其中Seq-2、Seq-3、Seq-6、Seq-19号核酸适配体能高特异性结合肺癌血清,与正常人血清不结合,再通过200例肺癌血清和200例正常人血清进一步鉴定4条肺癌血清核酸适配体检出肺癌的阳性率,阳性检出率达91%以上,为肺癌的早期诊断提供了新方法。

     

  • 图  1  实时定量-PCR检测肺癌血清核酸适配体富集结果

    图  2  肺癌血清核酸适配体亲和力检测结果

    图  3  肺癌血清核酸适配体同源性及进化树分析结果

    图  4  部分肺癌血清核酸适配体二级结构预测图

    图  5  肺癌血清核酸适配体特异性鉴定结果

    表  1  肺癌血清核酸适配体SELEX筛选条件

    轮次 磁珠量(mL) 收获ssDNA文库量(pmol) ssDNA文库量(pmol) 肺癌混合血清量(μL) 文库/磁珠结合血清 孵育时间(min)
    1 1 69.5 69.5 600 2 60
    2 1 46.3 46.3 300 2 60
    3 0.5 79.8 52 350 4 50
    4 0.5 59.7 50 300 4 50
    5 0.3 42.9 40 275 6 50
    6 0.25 51.6 45 250 6 40
    7 0.2 49.1 40 225 8 40
    8 0.15 30.1 30 200 8 40
    9 0.10 28.1 28 175 10 30
    下载: 导出CSV

    表  2  肺癌血清核酸适配体序列

    适配体 核酸序列 丰度(%) Kd(nmol/L)
    Seq-2 5′-CTATAGCAATGGTACGGTACTTCCCAATGGTACGGTACTTCCAA
    GCTAACCCTCATCTGCGCGCCAAAAGTGCACGCTACTTTGCTAA-3′
    16 22±7.2
    Seq-3 5′-CTATAGCAATGGTACGGTACTTCCCCTCGGCACGTTCTCAGTAG
    CGCTCGCTGGTCATCCCACACAAAAGTGCACGCTACTTTGCTAA-3′
    13 29±8.9
    Seq-6 5′-CTATAGCAATGGTACGGTACTTCCCCAAGCTAACCCCCATCTG
    CGCGCTCCTCAGTAGCGCTGCCAAAAGTGCACGCTACTTTGCTAA-3′
    15 35±6.3
    Seq-19 5′-CTATAGCAATGGTACGGTACTTCCATGGTACGGTACTTCCAAG
    CTAACCCCCATCTGCGCGCTCCAAAAGTGCACGCTACTTTGCTAA-3′
    20 15±8.6
    Seq-25 5′-CTATAGCAATGGTACGGTACTTCCAAGCTAACCCTCATCCGCG
    CGCTCCCCAAGCAGTATGCGCCAAAAGTGCACGCTACTTTGCTAA-3′
    4 76±8.4
    Seq-32 5′-CTATAGCAATGGTACGGTACTTCCTACGACTCACTATAGGAGCT
    AACCC TCATCTGCGCGCTCCCAAAAGTGCACGCTACTTTGCTAA-3′
    2 106±7.2
    Seq-51 5′-CTATAGCAATGGTACGGTACTTCCAAGCTGACCCTCATCTGCGC
    GCTC CACCCTCATCTACGCGCAAAAGTGCACGCTACTTTGCTAA-3′
    3.5 109±4.9
    Seq-54 5′-CTATAGCAATGGTACGGTACTTCCAAGCTAACCCTCATCTGCAC
    GTATCGGCTCTGGTAAGGTCCAAAAGTGCACGCTACTTTGCTAA-3′
    3 125±7.4
    Seq-69 5′-CTATAGCAATGGTACGGTACTTCCGGACGCGGTTAGGGGAGAA
    TTTCCATACCTTTGGGTTGTACAAAAGTGCACGCTACTTTGCTAA-3′
    1.5 99±4.6
    Seq-76 5′-CTATAGCAATGGTACGGTACTTCCCTTTGTGCGGCCTGCAGCCG
    TAGTAGTTGGTTTAACGCGTCAAAAGTGCACGCTACTTTGCTAA-3′
    1 89±6.9
    下载: 导出CSV
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  • 收稿日期:  2018-03-04
  • 刊出日期:  2018-07-01

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