CN110184273B - 特异性靶向乳腺癌细胞株mcf-7的核酸适配体筛选及其应用 - Google Patents
特异性靶向乳腺癌细胞株mcf-7的核酸适配体筛选及其应用 Download PDFInfo
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Abstract
本发明公开了一种特异性靶向乳腺癌细胞株MCF‑7的核酸适配体筛选及其应用，核酸适配体的核苷酸序列是指如下序列所示的DNA片段：序列9‑A：5’‑AGGAGCACGACTCTGACGTAGGATCGAGACGAGGTACGTAT‑3’，是通过cell‑SELEX技术进行筛选，首先合成初始文库，以乳腺癌细胞株MCF‑7为靶细胞，与文库孵育，进行正筛选、洗脱、PCR扩增和单链DNA制备。本发明的核酸适配体进行乳腺癌细胞检测时，操作简洁方便、成本低、周期短、准确性高等优点，利用本发明的核酸适配体对其结合的靶分子进行研究，可以得到其肿瘤标志物，这对于乳腺癌的早期诊断、乳腺癌分型分析等方面应用提供了一种有效的工具和手段，可用于乳腺癌的早期诊断、制备乳腺癌诊断药物及试剂盒等。
Description
技术领域
本发明涉及生物医学技术领域，具体是一种特异性靶向乳腺癌细胞株MCF-7的核酸适配体筛选及其应用。
背景技术
核酸适配体(aptamer)也被称作适配体、适配子等，是一类小分子RNA或单链DNA，通常由20-100个碱基组成，从人工合成的RNA/DNA文库中筛选分离出能与目标物质进行高特异性、高灵敏度结合的单链寡核苷酸。虽然适配体的结合方式与抗体较为相似，但核酸适配体特有的性质在很多方面都超过了抗体，被学者称为“化学抗体”。与抗体相比，核酸适配体完全通过体外筛选，在生产成本、稳定性、修饰难易程度和组织穿透力等方面具有明显优势。核酸适配体有望替代抗体，在分析化学、临床医学以及分子生物学等各个领域具有广泛的应用前景。
SELEX技术是研究适配体的通用手段和强大工具，但传统的SELEX技术筛选效率低下、筛选时间过长、过程繁杂。为了扩宽SELEX技术的使用范围，提高其筛选效率，发展出了不同的新型SELEX技术，如cell-SELEX技术。cell-SELEX技术在不需要知道靶标分子的情况下，对生理状态下的天然构象的靶蛋白能进行筛选，得到特异性适配体，且不影响细胞本身和其生物学功能。cell-SELEX技术具有以下优点：靶标是完整细胞，不需要体外合成和纯化，得到的适配体能结合天然构象下的生物分子，更加适用于医学临床；同时筛选分离出针对细胞表面多种生物分子的适配体；该技术针对的是靶细胞表面的差异分子，因此在筛选之前不需要对细胞表面分子有全面的了解。
乳腺癌是发生在乳腺腺上皮组织的恶性肿瘤，乳腺癌已成为全球女性最常见的恶性肿瘤。据统计，2018年全球新增乳腺癌病例约210万，死亡病例约63万，乳腺癌发病率和死亡率均居女性癌症之首。早发现、早诊断和早治疗仍然是提高患者生存期的最有效途径。因此，发展能高灵敏度、高特异性识别乳腺癌细胞的分子探针，对于实现乳腺癌的早期诊断、转移预警和疗效评估等具有重要意义。
发明内容
本发明的目的在于提供一种特异性靶向乳腺癌细胞株MCF-7的核酸适配体筛选及其应用，以解决上述背景技术中提出的问题。
为实现上述目的，本发明提供如下技术方案：
一种特异性靶向乳腺癌细胞株MCF-7的核酸适配体，核酸适配体的核苷酸序列是指如下序列所示的DNA片段：
序列9-A：5’-AGGAGCACGACTCTGACGTAGGATCGAGACGAGGTACGTAT-3’。
进一步的，上述的所述核酸适配体对用于人乳腺癌细胞株MCF-7的识别以及应用在制备早期诊断人乳腺癌试剂盒中。
另外，本发明还提供了一种上述所述的核酸适配体筛选方法，利用cell-SELEX技术，以人乳腺癌细胞株MCF-7为靶细胞，人乳腺正常细胞MCF-10A为对照细胞，进行乳腺癌细胞核酸适配体筛选，其具体步骤如下：
(1)以MCF-7细胞进行前3轮正向筛选，第4轮开始加入MCF-10A对照细胞进行反向筛选，筛选过程中逐步增大筛选压力以提高适配体次级文库对MCF-7细胞的高特异结合，每一轮均通过测量的荧光强度计算结合率，通过荧光倒置显微镜成像进行表征；
(2)观察筛选结果，选择核酸适配体次级文库富集最大的一轮：筛选结果显示第14轮筛选时核酸次级文库富集达到最大，将此单链DNA文库克隆于pEASY-T1载体中，并将成功克隆的30个菌落进行测序，获得能与乳腺癌细胞株MCF-7结合的20条不同的核酸适配体序列；
(3)利用DNAMAN软件对20条核酸适配体序列进行系统分析，对其中重复率最高的序列9进行二级结构预测和截短优化，通过全自动酶标仪和荧光倒置显微镜来对其亲和力进行考察，得到亲和力高、特异性强的核酸适配体9-A。
与现有技术相比，本发明的有益效果是：
1、本发明通过cell-SELEX技术筛选得到的核酸适配体，与传统以纯化蛋白或人工合成蛋白为靶点相比，具有可人工合成、结构简单、亲和力高、特异性强，能更好的与天然状态下的乳腺癌细胞株MCF-7进行结合。
2、本发明的核酸适配体进行乳腺癌细胞检测时，操作简洁方便、成本低、周期短、准确性高等优点，利用本发明的核酸适配体对其结合的靶分子进行研究，可以得到其肿瘤标志物，这对于乳腺癌的早期诊断、乳腺癌分型分析等方面应用提供了一种有效的工具和手段。
3、本发明的核酸适配体可用于乳腺癌的早期诊断、制备乳腺癌诊断药物及试剂盒等。
附图说明
图1是本发明实例中利用Valfold软件模拟的序列9、序列9-A、序列9-B、序列9-C的二级结构示意图。
图2是本发明中核酸适配体的结合能力检测结果。
图3是核酸适配体9-A、核酸适配体9-B、核酸适配体序列9的荧光显微图(a-核酸适配体9-A；b-核酸适配体9-B；c-核酸适配体序列9)。
图4是核酸适配体9-A的解离常数曲线拟合图。
图5是温度对核酸适配体与细胞结合能力的影响图
具体实施方式
下面将结合本发明实施例中的附图，对本发明实施例中的技术方案进行清楚、完整地描述，显然，所描述的实施例仅仅是本发明一部分实施例，而不是全部的实施例。基于本发明中的实施例，本领域普通技术人员在没有做出创造性劳动前提下所获得的所有其他实施例，都属于本发明保护的范围。
请参阅图1～4，本发明实施例中，一种特异性靶向乳腺癌细胞株MCF-7的核酸适配体筛选，利用cell-SELEX技术，以人乳腺癌细胞株MCF-7为靶细胞，人乳腺正常细胞MCF-10A为对照细胞，进行乳腺癌细胞核酸适配体筛选，其具体步骤如下：
(1)以MCF-7细胞进行前3轮正向筛选，第4轮开始加入MCF-10A对照细胞进行反向筛选，筛选过程中逐步增大筛选压力以提高适配体次级文库对MCF-7细胞的高特异结合，每一轮均通过测量的荧光强度计算结合率，通过荧光倒置显微镜成像进行表征；
(2)观察筛选结果，选择核酸适配体次级文库富集最大的一轮：筛选结果显示第14轮筛选时核酸次级文库富集达到最大，将此单链DNA文库克隆于pEASY-T1载体中，并将成功克隆的30个菌落进行测序，获得能与乳腺癌细胞株MCF-7结合的20条不同的核酸适配体序列；
(3)利用DNAMAN软件对20条核酸适配体序列进行系统分析，对其中重复率最高的序列9进行二级结构预测和截短优化(如图1、表1所示)，通过全自动酶标仪和荧光倒置显微镜来对其亲和力进行考察(如图2、图3所示)，得到亲和力高、特异性强的核酸适配体9-A，核心序列主要为茎环结构，其解离常数Kd值为113.47nM(如图4所示)，达到了纳摩尔水平。
表1-截短优化序列
另外，参考图5所示，在4℃、25℃、37℃及40℃四个不同温度下，9-A均能较好的与乳腺癌细胞MCF-7结合，具有较好的温度稳定性；9-A能特异性识别乳腺癌细胞MCF-7，而对MCF-10A乳腺细胞、HL-7702肝细胞、MRC-5肺细胞三种正常细胞，HepG-2肝癌细胞、A549肺癌细胞、SW620结肠癌细胞、CNE-2鼻咽癌细胞四种癌细胞没有识别作用(如表2所示)。
表2-核酸适配体的细胞特异性考察
本发明中筛选的核酸适配体可用于人乳腺癌细胞株MCF-7的识别以及应用在制备早期诊断人乳腺癌试剂盒中。
本发明通过cell-SELEX技术筛选得到的核酸适配体，与传统以纯化蛋白或人工合成蛋白为靶点相比，具有可人工合成、结构简单、亲和力高、特异性强，能更好的与天然状态下的乳腺癌细胞株MCF-7进行结合；
采用本发明的核酸适配体进行乳腺癌细胞检测时，操作简洁方便、成本低、周期短、准确性高，利用本发明的核酸适配体对其结合的靶分子进行研究，可以得到其肿瘤标志物，这对于乳腺癌的早期诊断、乳腺癌分型分析等方面应用提供了一种有效的工具和手段。
以上所述，仅为本发明较佳的具体实施方式，但本发明的保护范围并不局限于此，任何熟悉本技术领域的技术人员在本发明揭露的技术范围内，根据本发明的技术方案及其发明构思加以等同替换或改变，都应涵盖在本发明的保护范围之内。
Claims (2)
1.一种特异性靶向乳腺癌细胞株MCF-7的核酸适配体，其特征在于，核酸适配体的核苷酸序列是指如下序列所示的DNA片段：
序列9-A：5’—AGGAGCACGACTCTGACGTAGGATCGAGACGAGGTACGTAT—3’。
2.一种如权利要求1所述的核酸适配体，其特征在于，所述核酸适配体用于人乳腺癌细胞株MCF-7的识别以及应用在制备早期诊断人乳腺癌试剂盒中。
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