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CN111909933B - 靶向细胞表面抗原mfi2蛋白的核酸适配体及应用 - Google Patents

靶向细胞表面抗原mfi2蛋白的核酸适配体及应用 Download PDF

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CN111909933B
CN111909933B CN202010609312.8A CN202010609312A CN111909933B CN 111909933 B CN111909933 B CN 111909933B CN 202010609312 A CN202010609312 A CN 202010609312A CN 111909933 B CN111909933 B CN 111909933B
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mfi2
aptamer
cell surface
surface antigen
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那仁满都拉
杨畅
王云
洪德飞
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Zhejiang University ZJU
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Abstract

本发明提供一种靶向细胞表面抗原MFI2蛋白的核酸适配体及应用，其序列如SEQ NO.1所示。本发明采用细胞筛选的方法从ssDNA文库中筛选出靶向细胞表面抗原MFI2蛋白的适配体，并对筛选出的文库中的序列通过高通量测序的手段进行分析，最终通过对所得序列的稳定性以及亲和性的分析，挑选出适配体MS2‑1。并且通过流式细胞术和免疫荧光的方法证明适配体MS2‑1能够特异性地靶向MFI2阳性的肿瘤细胞并通过内吞作用进入细胞。可在制备胰腺癌靶向治疗药或检测试剂中的应用。

Description

靶向细胞表面抗原MFI2蛋白的核酸适配体及应用
技术领域
本发明属生物制药领域，具体涉及一种靶向细胞表面抗原MFI2蛋白的核酸适配体及应用。
背景技术
胰腺癌因其早期诊断困难、后期治疗效果差，易局部复发和远端转移，被认为是死亡率最高的恶性肿瘤之一。分子靶向治疗因对肿瘤细胞有高特异性及高效杀伤性而被认为是治疗胰腺癌的有效手段。在前期研究中，我们通过对胰腺癌患者组织样本膜蛋白的分析，证实了胰腺癌特异性膜蛋白—黑素转铁蛋白(Melanotransferrin，MFI2)是敏感性高、特异性强、结构稳定的胰腺癌治疗新靶点。
目前大多数抗肿瘤药物都对肿瘤细胞有很好的杀伤作用，但由于缺乏对肿瘤细胞的特异性识别能力，其药效大大减弱的同时对正常细胞也存在极大的毒副作用。而抗体偶联药物因其对肿瘤细胞具有高特异性及高效杀伤性而备受关注。但同时，抗体药物仍存在诸多不足之处，昂贵的价格，以及抗体大分子在体内造成的免疫原性而危及生命等等问题亟待解决。
核酸适配体(Aptamer)是一类具有高亲和力和特异性的单链DNA或RNA寡核苷酸序列，有着与抗体类似的精准靶向性，它能够通过自身折叠所形成的发夹、茎环等结构结合其靶标上的互补空间结构，实现特异性识别功能。同时，与单克隆抗体相比，核酸适配体制备简单，成本较低，并且其分子量小于抗体，不易发生免疫反应，同时单链DNA的化学稳定性好，变性与复性可逆，易于长期保存和室温运输，是理想的靶向分子。因此，将核酸适配体与抗肿瘤药物偶联将会是肿瘤的分子靶向治疗一重大突破。
发明内容
本发明的目的是提供一种靶向细胞表面抗原MFI2蛋白的核酸适配体，是一种用于靶向胰腺癌特异性抗原MFI2蛋白的单链DNA核酸适配体。
本发明提供的核酸适配体的序列如SEQ NO.1所示：CACACATGATGCAGATCATGCGTCGATACTGTGTGTGCCTATGCGTGCTACCGTGAA。
本发明的另一个目的是提供所述的核酸适配体在制备胰腺癌靶向治疗药或在制备胰腺癌的检测试剂中的应用。
本发明采用细胞筛选的方法从ssDNA文库中筛选出靶向胰腺癌特异性抗原MFI2蛋白的适配体，并对筛选出的文库中的序列通过高通量测序的手段进行分析，最终通过对所得序列的稳定性以及亲和性的分析，挑选出适配体MS2-1。并且通过流式细胞术和免疫荧光的方法证明适配体MS2-1能够特异性地靶向MFI2阳性细胞。
附图说明
图1是核酸适配体MS2-1对MFI2-/-Panc-1细胞的亲和性分析。
图2是核酸适配体MS2-1在MFI2-/-293T细胞及MFI2+/+Panc-1细胞中的特异性靶向及内吞分析。
图3是核酸适配体偶联药物MS2-1-Dox对MFI2+/+Panc-1细胞和MFI2-/-细胞NB4细胞的毒性分析。
具体实施方式
本发明结合附图和实施例作进一步的说明。
实施例1：适配体MS2-1与MFI2+/+Panc-1细胞的亲和性。
将293T和Panc-1细胞用binding buffer洗涤两次，EDTA消化后配制成单细胞悬液。计数后，50万/组加至离心管中重悬于binding buffer中。每管分别加入250nM DNA，并用binding buffer定容至100μl。37℃避光孵育30min，离心去除上清，并用binding buffer洗涤两次。加入500μl binding buffer，混匀，用流式细胞术检测。
通过流式细胞术检测并绘制亲和曲线，计算得到Kd值为39nM，说明核酸适配体MS2-1与Panc-1细胞有较好的亲和性，参见图1。
实施例2：适配体MS2-1在MFI2阳性细胞和MFI2阴性细胞内的内吞。
将提前种有293T与Panc-1细胞的小圆片从6孔板内夹出，4％多聚甲醛固定30min后，binding buffer洗涤两次。用binding buffer配置250nM的MS2适配体溶液，每个小圆片上滴加50ul，分别在4℃和37℃避光孵育0.5h、1h、3h。用binding buffer洗涤3次，加DAPI封片。
结果表明，4℃条件下，在孵育3h后核酸适配体MS2-1能与MFI2+/+Panc-1细胞膜表面结合，但不能被细胞内吞。37℃条件下，核酸适配体MS2-1在孵育30min后开始被Panc-1细胞内吞，而MFI2-/-293T细胞孵育3h后仍不能内吞，表明核酸适配体MS2-1能够特异性结合MFI2+/+Panc-1细胞并被内吞，对于MFI2-/-293T细胞则不能结合并被内吞，参见图2。
实施例3：适配体MS2-1与阿霉素偶联特异性抑制MFI2阳性胰腺癌细胞增殖。
将NB4细胞和Panc-1细胞种于96孔板中，每孔10000个细胞，加入10ul不同浓度的适配体MS2与阿霉素的偶联药物MS2-1-Dox或阿霉素单药，孵育72h后，加入20ul浓度为5mg/ml的MTT，4h后加入100ul的三联液，孵育16h后于595nm波长检测吸光度。
结果表明，核酸适配体MS2-1与阿霉素的偶联药物MS2-1-Dox与阿霉素单药类似能够特异性的抑制MFI2+/+Panc-1细胞的增殖，而对于MFI2-/-的NB4细胞，偶联药物无法抑制其增殖，能够显著减弱阿霉素的毒性，表明偶联药物MS2-1-Dox能够特异性地将阿霉素递送至MFI2阳性细胞中，参见图3。
序列表
<110> 浙江大学
<120> 靶向细胞表面抗原MFI2蛋白的核酸适配体及应用
<160> 1
<170> SIPOSequenceListing 1.0
<210> 1
<211> 57
<212> DNA
<213> 利用cell-SELEX技术筛选出的适配体序列(人工序列Unknown)
<400> 1
cacacatgat gcagatcatg cgtcgatact gtgtgtgcct atgcgtgcta ccgtgaa 57

Claims (3)

1.一种靶向细胞表面抗原MFI2蛋白的核酸适配体，该核酸适配体的序列如SEQ NO.1所示：
CACACATGATGCAGATCATGCGTCGATACTGTGTGTGCCTATGCGTGCTACCGTGAA。
2.根据权利要求1所述的一种靶向细胞表面抗原MFI2蛋白的核酸适配体在制备胰腺癌靶向治疗药中的应用。
3.根据权利要求1所述的一种靶向细胞表面抗原MFI2蛋白的核酸适配体在制备胰腺癌检测试剂中的应用。
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