CN110628770B - 靶向OFA/iLRP的核酸适配体 - Google Patents
靶向OFA/iLRP的核酸适配体 Download PDFInfo
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Abstract
本发明提供一种靶向OFA/iLRP的核酸适配体,包含如SEQ ID No.1所示核苷酸序列。该分子为能够结合OFA/iLRP的DNA核酸适配体。该核酸适配体能够以较高的亲和力与OFA/iLRP抗原结合,同时与其他蛋白如白蛋白、卵清蛋白、胰蛋白酶等结合较弱。该核酸适配体能够特异性结合OFA/iLRP阳性肿瘤细胞。此外,该核酸适配体具有较强的抵抗核酸酶的能力,具有较好的体内应用潜能。该核酸适配体有望成为OFA/iLRP阳性肿瘤细胞靶向治疗的配体分子。
Description
技术领域
本发明属于肿瘤的靶向治疗领域,涉及一种核酸适配体,具体涉及一种靶向OFA/iLRP的核酸适配体,通过将治疗肿瘤的药物靶向递送到肿瘤细胞,从而实现肿瘤的靶向杀伤。
背景技术
恶性肿瘤是一类可以浸润破坏器官结构和功能、发生转移,且以细胞分化不成熟和生长迅速为特征的疾病,是目前危害人类健康的最严重疾病之一。世界卫生组织在《全球癌症报告2015》中指出,全球癌症患者和死亡病例都在快速增加,2012年全球有1400万恶性肿瘤新发病例,其死亡人数占人类死亡总数的14.6%,且新发肿瘤超过50%来自中国。传统的肿瘤治疗方法主要包括外科手术治疗、放疗和化疗。但对于肿瘤发生转移的晚期患者,病人接受手术治疗的可能性降低,而化疗由于缺少肿瘤靶向性,在杀伤肿瘤细胞的同时也会对正常组织产生损伤,具有很强的毒副作用,从而限制了化疗的治疗剂量及频率,使得化疗难以清除机体内的全部肿瘤细胞,导致肿瘤复发或转移。因此,急需新的治疗方法来解决这一难题。靶向治疗,通过将抗肿瘤药物靶向递送到肿瘤细胞,使得药物更多地富集在肿瘤组织内,在杀伤肿瘤细胞的同时减少对机体正常组织细胞的损伤,从而有助于清除体内的肿瘤细胞,并大大降低毒副作用。靶向治疗成为一种新型抗肿瘤策略。
实现肿瘤靶向治疗,需要有在肿瘤细胞表面高表达的靶标。OFA/iLRP是分子量为37kDa的膜蛋白,在多种肿瘤细胞表面高表达,包括结肠癌、宫颈癌、卵巢癌、肺癌、纤维肉瘤、急性髓系白血病、淋巴瘤等。而在正常组织细胞中,OFA/iLRP仅在胚胎期及胎儿期表达于细胞表面,出生后消失,不在正常细胞表面表达。因此,OFA/iLRP有望成为一个良好的抗肿瘤靶点。
肿瘤靶向治疗还需要能特异性识别肿瘤靶标的配体,核酸适配体即为其中一种。核酸适配体是一种短链DNA或RNA分子,能够形成复杂的含有茎环、凸起、发夹、假结或G-四联体等的空间结构,从而与靶标特异性结合。它的靶标可以是蛋白质、多肽、核酸、氨基酸、细胞,甚至是一些金属离子等。此外,核酸适配体具有能够体外大量合成、价格便宜、稳定性好、便于储存等优点,有望为新的靶向治疗方法提供更好的策略。
核酸适配体应用于肿瘤靶向治疗,需要有较高的抵抗核酸酶的能力。因为体内存在大量的核酸酶,而普通的核酸适配体通常不能够抵抗核酸酶,在体内应用时很容易被核酸酶降解,从而失去结合靶标的能力。目前,以OFA/iLRP为靶标的具有抵抗核酸酶能力的核酸适配体尚未见报道。
发明内容
为了解决上述问题,本发明目的在于合成一种靶向OFA/iLRP的核酸适配体,该核酸适配体为一种新型核酸适配体,能够以较高的亲和力和特异性结合OFA/iLRP抗原,并且能够识别OFA/iLRP阳性的肿瘤细胞,同时与其他蛋白及阴性细胞的交叉结合较弱。此外,该适配体具有良好的抵抗核酸酶的能力,具有较好的体内应用潜能。为将来通过该核酸适配体将抗肿瘤药物靶向递送到OFA/iLRP阳性肿瘤细胞,并降低机体正常组织细胞对药物的摄取,从而实现肿瘤细胞的靶向杀伤提供实验基础。
为了实现上述目的,本发明提供一种靶向OFA/iLRP的核酸适配体,包含如SEQ IDNo.1所示核苷酸序列:5’-GTTGTTTGTATTGTTGTCTATCCTCTTAGGGATTT-3’。
本发明还提供上述的靶向OFA/iLRP的核酸适配体在作为用于检测和/或治疗OFA/iLRP阳性的肿瘤细胞的药物中的应用。
其中,所述OFA/iLRP阳性的肿瘤细胞为结肠癌细胞、宫颈癌细胞、卵巢癌细胞、肺癌细胞、纤维肉瘤细胞、急性髓系白血病细胞、淋巴瘤细胞。
本发明的有益效果在于:
本发明提供一种靶向OFA/iLRP的核酸适配体,该核酸适配体通过化学合成的方法得到了一个全新的分子结构,即以OFA/iLRP为靶标的核酸适配体,该分子为含有35个碱基的单链DNA。该分子能够自发形成空间结构,以较高的亲和力(75.86nM)及特异性识别靶标分子OFA/iLRP,并且能够与OFA/iLRP阳性肿瘤细胞结合,同时与阴性蛋白及阴性肿瘤细胞交叉结合较弱。此外,该分子具有较强的抵抗核酸酶的能力。由于血清中存在大量的核酸酶,将该分子置于50%血清中处理24h,仍有大量分子未被降解。且在20%血清条件下仍然能够与OFA/iLRP阳性肿瘤细胞结合,显示出了较好的体内应用前景。
附图说明
图1为本发明提供的靶向OFA/iLRP的核酸适配体的特异性检测结果图。
图2为本发明提供的靶向OFA/iLRP的核酸适配体亲和常数的测定结果。
图3为本发明所提供的靶向OFA/iLRP核酸适配体与OFA/iLRP阳性细胞、OFA/iLRP阴性细胞的结合的检测结果图。
图4为本发明所提供的靶向OFA/iLRP核酸适配体的抗核酸酶能力的检测结果图。
图5为本发明所提供的靶向OFA/iLRP核酸适配体在20%FBS条件下与OFA/iLRP阳性细胞、OFA/iLRP阴性细胞的结合的检测结果图。
具体实施方式
近年来,肿瘤靶向治疗越来越受到关注,因为与传统的放、化疗相比,靶向治疗在发挥药效的同时,还可减轻药物对机体正常组织、细胞的损伤。OFA/iLRP是高表达于多种肿瘤细胞表面的分子,是肿瘤治疗的重要靶点。靶向治疗需要靶向分子,核酸适配体(Aptamer)是一种具有较大应用潜能的新型靶向分子。核酸适配体是由短链DNA或RNA构成,可以通过自身折叠形成复杂的空间结构,以较高的亲和力及特异性与靶标结合。与抗体相比,核酸适配体具有价格低廉、易于合成、易于修饰、不同批次间质量较为稳定等优势,有望成为一种相对理想的靶向分子。
一、材料与方法
1.核酸适配体及同等长度的随机单链DNA由Invitrogen公司合成。
2.FITC标记的核酸适配体及同等长度的随机DNA由Invitrogen公司合成。
3.OFA/iLRP抗原购买自上海波泰生物科技有限公司。
4.牛血清白蛋白购买自天津灏洋生物科技有限公司。
5.卵清蛋白购买自Amresco公司。
6.胰蛋白酶购买自Amresco公司。
7.链霉亲和素包被磁珠(单分散磁珠):购买自普洛麦格公司(磁珠表面为链霉亲和素修饰)。
8.环氧基修饰的磁性微球(磁珠):Affimag UF,表面环氧基修饰,购买自天津倍思乐色谱技术开发中心。
9.细胞系:人早幼粒急性白血病细胞HL-60,人B细胞淋巴瘤细胞Ramos,人T细胞淋巴瘤细胞Jurket均购买自中国医学科学院细胞中心。
10.PBMC:健康志愿者外周血提取。
11.细胞培养基RPMI1640购买自中国医学科学院基础医学研究所细胞中心。
12.胎牛血清(FBS)购买自Gibico公司。
13.青霉素、链霉素购买自中国医学科学院基础医学研究所细胞中心。
14.流式细胞仪Accuri C6购买自中国医学科学院基础医学研究所中心实验室。
15.碳酸盐缓冲液(CBS):先分别配制0.2mol/L的碳酸钠溶液和0.2mol/L的碳酸氢钠溶液,然后将38.5mL碳酸钠溶液和11.5mL碳酸氢钠溶液混匀,即为pH=10.4的碳酸盐缓冲液。
实施例1.OFA/iLRP核酸适配体的特异性的测定
靶标和磁珠的连接:用于检测的靶标为OFA/iLRP抗原,抗原用ddH2O溶解,浓度为0.5μg/μL。
首先,取5×105个环氧基修饰的磁性微球,用PBS清洗3遍,然后加入500μL碳酸盐缓冲液(CBS,pH=10.4),重悬该磁性微球,将2μg OFA/iLRP抗原与该磁性微球混合,室温震荡孵育12h,PBS洗涤3次,再以PBS重悬该磁性微球,制成OFA/iLRP抗原包被的磁性微球,4℃保存备用。同样的方法制备牛血清白蛋白(BSA)、卵清蛋白(OVA)、胰蛋白酶(Trypsin)包被的磁性微球。
流式细胞仪分析:为了检测核酸适配体与靶标结合的特异性,将FITC标记的核酸适配体在200μL PBS缓冲液中分别与进行OFA/iLRP抗原包被的磁性微球、BSA包被的磁性微球、OVA包被的磁性微球或Trypsin包被的磁性微球于37℃震荡孵育30min,PBS洗3遍,200μLPBS重悬上述磁性微球,进行流式细胞仪分析。FITC标记的同等长度的随机DNA作为随机对照。
结果如图1所示,其中A为核酸适配体与OFA/iLRP抗原结合流式图,B为核酸适配体与白蛋白(BSA)结合流式图,C为核酸适配体与卵清蛋白(OVA)结合流式图,D为核酸适配体与胰蛋白酶(Trypsin)结合流式图,图中黑色曲线表示随机单链DNA分别与OFA/iLRP抗原、白蛋白、卵清蛋白、胰蛋白酶结合产生的荧光信号,灰色曲线表示OFA/iLRP核酸适配体分别与OFA/iLRP抗原、白蛋白、卵清蛋白、胰蛋白酶结合产生的荧光信号。
与随机对照相比,核酸适配体与BSA、OVA、Trypsin结合后荧光信号极弱(图1中B、C、D),而与OFA/iLRP抗原反应后荧光信号显著增强(图1中A),上述图1中A至D的横坐标为荧光强度(Fluorenscence intensity),纵坐标为磁珠数量(Events),DNA上有荧光修饰,如果DNA与磁性微球上的蛋白结合,流式仪检测得到的峰会右移,右移程度越大,说明结合到磁珠上蛋白的DNA越多。以随机DNA产生的峰为对照,如果核酸适配体产生的峰显著右移,说明核酸适配体与蛋白结合较强,如果两个峰无显著差异,就说明核酸适配体与蛋白结合很弱。在OFA/iLRP这一组,核酸适配体产生的峰显著右移,说明核酸适配体与靶标的结合显著强于随机DNA;而在其它蛋白组,这两种DNA的峰没有显著差异,说明核酸适配体几乎不结合这三种蛋白。说明本发明提供的核酸适配体能够特异性地结合OFA/iLRP,而与BSA、OVA、Trypsin等阴性蛋白结合很弱。说明该核酸适配体的特异性好。
实施例2OFA/iLRP核酸适配体亲和常数的测定
Kd值的测定:将实施例1中靶标OFA/iLRP包被的磁性微球(1×105个)分别与不同浓度(40、60、80、100、120、140、150、200、250、300nM)的FITC标记的核酸适配体在200μL结合缓冲液(PBS)中于37℃反应30min,PBS洗3遍,流式细胞仪检测平均荧光强度。根据公式Y=Bmax X/(Kd+X)(Y:平均荧光强度,X:所用的aptamer的浓度,B为一个常数,max是最大值的意思),计算核酸适配体和靶标结合的Kd值。
结果如图2所示(横坐标为核酸适配体浓度(Concentration),纵坐标为荧光强度(Fluorenscence intensity)),得出核酸适配体与靶标的Kd值为75.86nM,结果表明,该核酸适配体与靶标具有较好的亲和力。
实施例3OFA/iLRP核酸适配体与OFA/iLRP阳性肿瘤细胞、OFA/iLRP阴性细胞的结合
将人早幼粒急性白血病细胞HL-60、人T细胞淋巴瘤细胞Jurkat、人B细胞淋巴瘤细胞Ramos,分别培养在含有10%FBS、常规浓度青霉素(100U/mL)和链霉素(100ug/mL)的RPMI1640培养基中,置细胞于37℃、5%CO2培养箱中培养,所有试验所用细胞均是处于对数生长期的细胞。PBMC从健康志愿者外周血分离得到。分别收集2×105的HL-60、Jurkat、Ramos和PBMC细胞,将其和60pmol FITC标记的核酸适配体(5’-GTTGTTTGTATTGTTGTCTATCCTCTTAGGGATTT-3’)在200μL PBS中反应30min,PBS洗2遍,用流式细胞仪分析;FITC标记的同等长度的随机DNA作为随机对照。
结果如图3的A至D所示,横坐标为荧光强度(Fluorenscence intensity),纵坐标为细胞数量(Events),图中黑色曲线表示随机单链DNA分别与OFA/iLRP阳性细胞HL-60、Jurkat和Ramos以及与OFA/iLRP阴性细胞PBMC孵育产生的对照荧光信号,灰色曲线表示的核酸适配体和OFA/iLRP阳性细胞HL-60(图3的A)、核酸适配体和Jurkat(图3的B)及核酸适配体和Ramos(图3的C)孵育后的荧光强度显著高于随机对照,而与OFA/iLRP阴性细胞PBMC(图3的D)孵育后荧光强度与随机对照相似。上述实验表明,OFA/iLRP核酸适配体能够特异性地结合OFA/iLRP阳性肿瘤细胞,几乎不与OFA/iLRP阴性细胞结合,说明该核酸适配体有望成为OFA/iLRP新型靶向配体,为靶向OFA/iLRP的抗肿瘤治疗提供实验基础。
实施例4核酸适配体抗核酸酶能力的检测
用50%FBS或PBS将该核酸适配体配成浓度为100ng/μL的溶液,分别于37度孵育0、4、8、12、24、36h,95℃加热10min,降至室温后分别取10μL DNA进行琼脂糖凝胶电泳。
DNA凝胶电泳结果如图4所示,图4的A为50%FBS条件下DNA电泳图,图4的B为PBS条件下DNA电泳图。由于血清中存在大量的核酸酶,而该核酸适配体在50%FBS条件下处理24h仍有大量DNA未被降解,而一般的DNA不能抵抗核酸酶,24小时内基本就都被降解了,这说明该核酸适配体具有较强的抗核酸酶能力。此外,该核酸适配体在PBS条件下十分稳定,处理36h后DNA量几乎没有发生变化。上述结果表明,该核酸适配体结构较为稳定,具有较强的抗核酸酶能力,具有较好的体内应用前景。
实施例5 20%FBS条件下核酸适配体与OFA/iLRP阳性肿瘤细胞的结合
将人早幼粒急性白血病细胞HL-60、人T细胞淋巴瘤细胞Jurkat、人B细胞淋巴瘤细胞Ramos,分别培养在含有10%FBS、常规浓度青霉素和链霉素的RPMI1640培养基中,置细胞于37℃、5%CO2培养箱中培养,所有试验所用细胞均是处于对数生长期的细胞。PBMC从健康志愿者外周血分离得到。分别收集2×105的HL-60、Jurkat、Ramos和PBMC细胞,将其和60pmol FITC标记的核酸适配体在200μL含20%FBS的PBS中反应30min,用含20%FBS的PBS洗2遍,用200μL含20%FBS的PBS重悬细胞,流式细胞仪检测各种荧光信号。FITC标记的同等长度的随机DNA作为随机对照。
结果如图5的A至D所示,横坐标为荧光强度(Fluorenscence intensity),纵坐标为细胞数量(Events),图中黑色曲线表示随机单链DNA分别与OFA/iLRP阳性细胞HL-60、Jurkat和Ramos以及与OFA/iLRP阴性细胞PBMC孵育产生的荧光信号;灰色曲线表示核酸适配体分别与OFA/iLRP阳性细胞HL-60、Jurkat和Ramos以及与OFA/iLRP阴性细胞PBMC孵育产生的荧光信号。核酸适配体和OFA/iLRP阳性细胞HL-60(图5的A)、Jurkat(图5的B)及Ramos(图5的C)孵育后的荧光强度显著高于随机对照,而与OFA/iLRP阴性细胞PBMC(图5的D)孵育后荧光强度与随机对照相似。上述实验表明,在核酸酶存在的条件下,OFA/iLRP核酸适配体仍然能够特异性地结合OFA/iLRP阳性肿瘤细胞,而与OFA/iLRP阴性细胞结合很弱,说明该核酸适配体有望应用于肿瘤靶向治疗。
从上述实施例可以看出,本发明提供的靶向OFA/iLRP的核酸适配体,具有较高的亲和力和特异性,与OFA/iLRP抗体相比较,具有能够大量合成,价格低廉,易于修饰,易于保存,免疫原性低等优点,有望为新的靶向治疗方法提供更好的策略。
本发明通过DNA合成仪得到了一个全新的分子结构,该分子为能够结合OFA/iLRP的DNA核酸适配体。该核酸适配体能够以较高的亲和力与OFA/iLRP抗原结合,同时与其他蛋白如白蛋白、卵清蛋白、胰蛋白酶等结合较弱。该核酸适配体能够特异性结合OFA/iLRP阳性肿瘤细胞。此外,该核酸适配体具有较强的抵抗核酸酶的能力,具有较好的体内应用潜能。该核酸适配体有望成为OFA/iLRP阳性肿瘤细胞靶向治疗的配体分子。
SEQUENCE LISTING
<110> 中国医学科学院基础医学研究所
<120> 靶向OFA/iLRP的核酸适配体
<160> 1
<170> PatentIn version 3.3
<210> 1
<211> 35
<212> DNA
<213> 人工序列
<400> 1
gttgtttgta ttgttgtcta tcctcttagg gattt 35
Claims (3)
1.一种靶向OFA/iLRP的核酸适配体,其特征在于,为如SEQ ID No.1所示核苷酸序列。
2.如权利要求1所述的靶向OFA/iLRP的核酸适配体作为制备检测和/或治疗OFA/iLRP阳性的肿瘤细胞药物的应用。
3.如权利要求2所述的应用,其特征在于,所述OFA/iLRP阳性的肿瘤细胞为结肠癌细胞、宫颈癌细胞、卵巢癌细胞、肺癌细胞、纤维肉瘤细胞、急性髓系白血病细胞或淋巴瘤细胞。
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