CN105802973B - 一种靶向髓系分化抗原cd33蛋白的核酸适配体及应用 - Google Patents
一种靶向髓系分化抗原cd33蛋白的核酸适配体及应用 Download PDFInfo
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Abstract
本发明提供一种用于靶向髓系分化抗原CD33的单链DNA核酸适配体,其序列如SEQ NO.1所示。本发明采用细胞筛选的方法从ssDNA文库中筛选出靶向髓系分化抗原CD33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S30。并且通过流式细胞术和免疫荧光的方法证明适配体S30能够特异性地靶向CD33阳性细胞。可在制备急性髓性白血病靶向治疗药或急性髓性白血病的检测试剂中的应用。
Description
技术领域
本发明属生物制药领域,具体涉及一种靶向髓系分化抗原CD33蛋白的核酸适配体及其应用。
背景技术
细胞分化抗原CD33蛋白是一种跨膜受体,在大多数成年和儿童急性髓性白血病(AML)患者细胞中大量表达,是临床上用于抗体靶向治疗AML的特异性结合位点。研究表明,CD33多存在于AML患者髓母细胞中,而造血干细胞中不表达,因此为AML的治疗提供了一个理想靶标。
核酸适配体(又称化学抗体),具有精准的靶向性。核酸适配体能够快速而有效地分辨出靶分子结构上的细微差别,仅识别与其互补的空间结构,并与之结合,但其本身对肿瘤细胞不具备较强的杀伤力。另外,我国已发现近几百种具有抗肿瘤活性的海洋毒素,但由于其毒性反应过强而使药物开发中断。核酸适配体与海洋毒素偶联药物,即毒性药物与核酸适配体相连接组成靶向适配体偶联物,可以将药物“精确”地运送到靶细胞,既有效地提高了肿瘤局部的药物浓度,又可极大地降低体内其它组织、器官的毒性,从而达到真正靶向增效减毒的作用。
与研究热点单克隆抗体相比,“核酸适配体”拥有相似的靶向载体功能,但在制备和化学性质方面具有其独特的优势,更方便于实际应用:1)体外筛选,可快速人工合成;2)靶标类型比抗体广泛,蛋白质、病毒颗粒或完整细胞等均可作为筛选靶标;3)分子量小于抗体,不发生免疫反应,易于靶分子特异性结合,且解离常数可以达到pmol至nmol,其亲和力甚至高于天然配体;4)化学稳定性好,变性与复性可逆,易于长期保存和室温运输等优势。
发明内容
本发明的目的是提供一种用于靶向髓系分化抗原CD33蛋白的核酸适配体,其序列如SEQ NO.1所示:TACCAGTGCGATGCTCAGCACGCTTATAGGGGCTGGACAAAATTCTACCCAGCCTTTTCTGACGCATTCGGTTGAC。
本发明另一个目的是提供所述的核酸适配体在制备急性髓性白血病靶向治疗药或在制备急性髓性白血病检测试剂中的应用。
本发明采用细胞筛选的方法从ssDNA文库中筛选出靶向髓系分化抗原CD33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S30。并且通过流式细胞术和免疫荧光的方法证明适配体S30能够特异性地靶向CD33阳性细胞。最终适配体可用于制备急性髓性白血病的靶向治疗药,可降低化疗毒性,也可用于制备急性髓性白血病的检测试剂。
附图说明
图1是适配体S30与HL60细胞的亲和性分析。
图2是适配体S30在HL60细胞与Jurkat细胞内的内吞。
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:靶向髓系分化抗原CD33核酸适配体的筛选
阴性筛选(反筛)
待六孔板中HEK-293T细胞生长至90%,将第一个孔上层培养基吸出,用结合缓冲液洗涤两次;再加入预处理过的ssDNA文库,置于4℃冰箱内的水平摇床上,4℃、15rpm孵育30min。然后,将ssDNA文库从上一个孔吸出,加入预先洗涤两次的第二个孔中,4℃、15rpm孵育30min。方法同上,直至阴性筛选六轮后,吸出待用。
阳性筛选(正筛)
HEK-293T细胞瞬转CD33,48h后作为阳性细胞,用结合缓冲液洗涤细胞两次;将阴性筛选六轮后的ssDNA文库缓慢加入阳性细胞中,4℃、15rpm孵育30min。孵育完毕后,用结合缓冲液,缓慢洗涤细胞三次,再加入无DNA酶水500μl,用细胞刮刀将细胞收集入1.5ml离心管中。95℃加热10min重悬ssDNA,再13000g离心5min收集筛选后的ssDNA文库。PCR扩增、酶切、纯化后进行下一轮筛选。
经过两轮筛选后,利用高通量测序、计算机模拟分析的方法得到靶向髓系分化抗原CD33蛋白的适配体S30,其序列如SEQ NO.1所示:TACCAGTGCGATGCTCAGCACGCTTATAGGGGCTGGACAAAATTCTACCCAGCCTTTTCTGACGCATTCGGTTGAC。
实施例2:适配体S30与CD33阳性细胞HL60结合。
取对数生长期HL60细胞,离心收集并用binding buffer洗涤细胞1次,用配制成单细胞悬液,计数后,50万/组加至离心管中重悬于binding buffer中。每管分别加入250nMDNA,并用binding buffer定容至100μl。其中,阴性对照组细胞不加任何处理。轻轻混匀,37℃避光孵育30min。离心去除上清,并用binding buffer洗涤两次。加入400μl bindingbuffer,混匀,上机检测。
结果表示,核酸适配体S30与HL60细胞有较好的亲和性,且用流式细胞术测核酸适配体的亲和性曲线得出,S30的Kd值为46nM,参见图1。
实施例3:适配体S30在CD33阳性细胞和CD33阴性细胞内的内吞。
取对数生长期HL60与Jurkat细胞,离心收集并用binding buffer洗涤细胞1次,用配制成单细胞悬液,计数后,15万/组加至96孔板中重悬于binding buffer中。每管分别加入250nM DNA,并用binding buffer定容至100μl。轻轻混匀,37℃避光孵育0.5h、1h、3h。离心去除上清,并用binding buffer洗涤一次,细胞甩片机甩片,4%多聚甲醛固定30min,用binding buffer洗涤两次,加DAPI封片。
结果表明,核酸适配体S30在孵育30min后开始被HL60细胞内吞,而Jurkat细胞孵育1h后仍不能内吞适配体S30,表明适配体S30能够特异性结合CD33阳性细胞HL60并被内吞,对于CD33阴性细胞Jurkat细胞则不能结合并被内吞,参见图2。
Claims (2)
1.一种靶向髓系分化抗原CD33蛋白的核酸适配体在制备急性髓性白血病靶向治疗药物载体中的应用,所述核酸适配体的序列如SEQ NO.1所示:
TACCAGTGCGATGCTCAGCACGCTTATAGGGGCTGGACAAAATTCTACCCAGCCTTTTCTGACGCATTCGGTTGAC。
2.一种靶向髓系分化抗原CD33蛋白的核酸适配体在制备急性髓性白血病的检测试剂中的应用,所述核酸适配体的序列如SEQ NO.1所示:
TACCAGTGCGATGCTCAGCACGCTTATAGGGGCTGGACAAAATTCTACCCAGCCTTTTCTGACGCATTCGGTTGAC。
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急性髓系白血病CD33+/CD34+细胞表面核酸适配体的筛选及结构分析;张书芹等;《中国实验血液学杂志》;20110914;第19卷(第3期);第561-565页 |
急性髓系白血病细胞单链DNA适体的体外筛选;朱平等;《中南大学学报(医学版)》;20121128;第37卷(第8期);第771-776页 |
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