CN107083388B - 一种特异结合膜联蛋白A2的核酸适体wh3及用途 - Google Patents
一种特异结合膜联蛋白A2的核酸适体wh3及用途 Download PDFInfo
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Abstract
本发明公开了特异结合膜联蛋白A2(Annexin A2,ANXA2）的核酸适体wh3及其应用，wh3序列如SEQ ID NO.3所示，该核酸适体能够与ANXA2特异结合，因此可以用于ANXA2的纯化和浓缩，也可以与治疗药物结合后作为靶向ANXA2的药物,进一步研究表明wh3能阻断ANXA2的作用，抑制多发性骨髓瘤（Multiple Myeloma，MM）细胞的生长和粘附且可作为ANXA2定量或定性检测试剂，应用前景广泛。
Description
技术领域
本发明属于适体领域，具体涉及特异结合膜联蛋白A2的核酸适体，还涉及该核酸适体的应用。
背景技术
膜联蛋白A2(Annexin A2，ANXA2)是由339个氨基酸组成的分子量为36 kDa的蛋白质, 是一种Ca2+依赖性磷脂结合蛋白，在细胞中可以单体(p36)、异源二聚体(p36/p11)和异源四聚体(p362/p112)三种形式存在。它在结构上高度保守并且在几乎所有真核生物中表达，主要分布在细胞膜、细胞浆中，一小部分存在于细胞核中。ANXA2 主要表达于内皮细胞、单核细胞、巨噬细胞、神经细胞和某些肿瘤细胞中，在脑癌、肝癌、胰腺癌、乳腺癌、肺癌和结肠癌以及血液肿瘤中表达上调。ANXA2在肿瘤中的高表达与肿瘤的发生、发展、浸润、转移及预后密切相关。ANXA2在高侵袭、高转移的恶性肿瘤中高表达，提示ANXA2有望成为判断肿瘤浸润和转移能力及肿瘤病人预后的标志物，可作为肿瘤治疗潜在的靶分子，该核酸适体可用于肿瘤早期检测及治疗。
目前，ANXA2的检测方法主要有免疫组化法，酶联免疫法等免疫学检测法。依赖抗体的免疫学检测法操作简单，灵敏度高，无需大型昂贵仪器设备，适用于大量样品的检测，但是这种方法检测的假阳性率比较高，定量也不够准确。并且检测所用抗体等试剂稳定性差，储存条件较DNA核酸适体严格。核酸适体能与各种有机物或无机物靶标分子高特异性、高亲和力结合，同时具有分子量小，免疫原性低，稳定性好，制备和标记方便等优点。核酸适体作为抗体分子的替代分子，在疾病的诊断和治疗中发挥重要作用。但迄今尚无针对ANXA2的DNA核酸适体报道或公开，因此有必要开发一种可特异结合ANXA2的核酸适体。
发明内容
有鉴于此，本发明目的在于提供特异结合并作用ANXA2的核酸适体。
本发明人采用消减筛选的策略，利用GST-ANXA2融合蛋白作为筛选靶标，可获得与融合蛋白结合的DNA序列，通过将GST蛋白作为负性筛选，将与GST蛋白结合的DNA序列去除，从而得到能与ANXA2蛋白特异结合的核酸适体，通过筛选获得了一条特异结合ANXA2的核酸适体（命名为wh3）。所述核酸适体的核苷酸序列如SEQ ID NO.3所示。
本发明提供所述特异结合ANXA2的核酸适体wh3在制备检测ANXA2试剂中的应用。具体地，通过将ANXA2蛋白与酶标板结合，然后利用生物素标记的wh3核酸适体孵育结合；PBS洗板后，加入过氧化物酶标记的链霉亲和素孵育，加入过氧化物酶的底物显色，通过酶标仪检测并计算ANXA2的浓度。
本发明提供特异结合ANXA2的核酸适体wh3在纯化和浓缩ANXA2中的应用。具体地，ANXA2蛋白与生物素标记的wh3核酸适体结合，然后加入链霉亲和素磁珠孵育，通过磁粉可将ANXA2纯化和浓缩。
本发明提供特异结合ANXA2的核酸适体在制备靶向ANXA2药物中的应用。具体而言，细胞膜表面的ANXA2蛋白能与多种蛋白结合发挥其生理作用，，wh3能与ANXA2蛋白结合，可能阻断ANXA2蛋白与其它蛋白的相互作用，其功能表现为wh3阻断ANXA2蛋白促进MM.1S和RPMI-8226细胞的生长，wh3能抑制MM1.S和RPMI-8226细胞对ANXA2蛋白的粘附作用。
本发明的有益效果在于：
本发明公开了特异结合ANXA2的核酸适体，该核酸适体能够与ANXA2特异性结合，为ANXA2纯化和浓缩，靶向ANXA2的药物以及定量或定性检测ANXA2提供了靶分子，同时为检测血清中ANXA2的水平，反映机体肿瘤负荷状态提供了工具，也为临床选择最佳的治疗方案提供了策略，为观察治疗效果和判断预后提供帮助。
附图说明
图1. wh3核酸适体的空间结构；
图2. ELISA检测wh3与ANXA2蛋白结合；
图3. Aptamer-pulldown检测wh3与ANXA2蛋白结合；
图4. wh3抑制ANXA2刺激MM.1S和RPMI-8226细胞的生长；
图5. wh3抑制HS-5细胞刺激MM.1S和RPMI-8226细胞的生长；
图6. wh3抑制MM.1S和RPMI-8226细胞与ANXA2蛋白的粘附。
具体实施方式
实施例1
合成长度为80 nt的核苷酸文库序列（上海生工生物工程（上海）股份有限公司），具体序列如下：5'-ACCGAC CGT GCT GGA CTC A (N)42 A CTA TGA GCGAGC CTG GCG-3'，两端为固定序列，本文库中间42N代表42个随机碱基，保证其库容量大约为1014，足够大库容量可形成不同的三维空间结构，从而保证具有与靶标结合的空间结构的序列存在，并在后面的筛选过程中筛选出来，库序列为P80 ss库。然后将随机寡聚ssDNA文库用如下引物进行扩增：
P80-SF：5'-FAM-ACC GACCGT GCT GGA CTC A-3' （SEQ ID NO.1）
P80-AP: 5'-biotin-CGC CAG GCT CGC TCA TAG T-3' （SEQ ID NO.2）
随机ssDNA文库（首轮筛选量为5000pmol，相当于4个OD随机库）用1mL 1 ×选择缓冲液溶解于EP管中（1%BSA包被，4℃封闭过夜），95℃变性10 min，变性后立即置于0℃放置10 min。将包被了GST蛋白的磁珠混匀，取100μL置于BSA包被的EP管中，用150μL结合缓冲液洗涤3次。将ssDNA文库（5 nmol）与包被了GST蛋白的磁珠混匀，室温孵育1 h，去除与GST蛋白磁珠结合的ssDNA序列。取上清ssDNA文库与包被了ANXA2磁珠混匀孵育1 h，同时加入酵母tRNA竞争结合。将EP管置磁力架上2-3 min，弃上清，用1×洗涤缓冲液洗去未结合的ssDNA序列,每次3 min，重复3次。置磁力架2-3 min，弃上清。EP管中加入200μL去离子水，95℃加热5 min，置磁力架2 min，取上清为PCR的模板，进行PCR扩增。
配置PCR体系：模板2μL，引物P80-SF（10μM）1μL，引物P80-AP（10μM）1μL，dNTP（各2.5 mM）4μL，10×buffer 5μL，Taq DNA聚合酶 0.25μL，ddH2O 36.75μL(总体积为50μL)。按如下条件，在PCR仪上扩增：95℃预变性3 min，95℃变性30 s，58℃退火30 s，72℃延伸30s，共25个循环，72℃延伸5 min。扩增后将PCR产物转移到1.5 mL的离心管中。用200μL 1×磁珠结合洗涤缓冲液重悬链霉亲和素琼脂糖珠，使链霉亲和素琼脂糖珠的终浓度为5μg/μL,加入1 mLPCR产物，室温摇床孵育结合1 h，然后1000 rpm，离心2 min，弃上清。用1×磁珠结合洗涤缓冲液洗涤链霉亲和素琼脂糖珠，每次5 min，重复3次。去上清，加入400μL的200mM NaOH与链霉亲和素琼脂糖珠孵育5 min，使dsDNA解链，然后1000 rpm，离心2 min，含生物素的一条ssDNA仍留在链霉亲和素琼脂糖珠上，而另一条不带生物素的ssDNA存在于上清中。分离上清中ssDNA即为下一轮筛选的富集库。重复进行9个循环筛选。
克隆测序及序列分析
将第9轮筛选得到的核酸适体富集库用无修饰的引物扩增，送上海生工生物工程有限公司进行高通量测序，测序成功后获得了两条与ANXA2结合的核酸适体，其中一条序列如SEQ ID NO.3所示，命名为wh3（另一条命名为wh6，另案予以申请）。具体序列如下：
ANXA2核酸适体(wh3)为：5'-ACCGACCGTGCTGGACTCAGGCCGATTGACTTTCCAACAAACTTAGGCCCACTAGACAGAAACTATGAGCGAGCCTGGCG-3' (SEQ ID NO.3)
然后通过mfold软件对wh3核酸适体序列的二级结构进行分析，结构如图1.所示。
实施例2
1×107个ANBL-6细胞用Western blot及IP细胞裂解液(碧云天，P0013)冰上裂解30 min，提取细胞总蛋白,取20μL蛋白液作为input。平均分到三个EP管中，分别加入50pmol的5’端生物素修饰的wh3核酸适体和DNA-Lib,并设链霉亲和素琼脂糖珠的阴性对照组,4℃摇床孵育1 h。然后加入20μL的链霉亲和素琼脂糖珠，4℃摇床孵育1 h。1000 rpm，离心2 min，弃上清，加入1mL的DPBS洗5 min，1000 rpm，离心2 min，弃上清。重复用DPBS洗三次，加入40μL的Western blot上样缓冲液到EP管中，95℃加热5 min。1000 rpm，离心2 min，取上清,SDS-PAGE电泳，转膜，牛奶封闭后用小鼠单克隆ANXA2抗体4℃孵育过夜，TPBS洗三次，加入HRP标记的兔抗鼠的IgG，通过ECL反应液显影，检测wh3核酸适体和DNA-Lib与ANXA2的结合情况。
结果显示（参见图2），DNAl-lib和链霉亲和素琼脂糖珠不与ANXA2蛋白结合，wh3核酸适体能与ANXA2蛋白结合。
实施例3
ANXA2重组蛋白用结合缓冲液稀释到1μg/mL,取200μL加入到酶标板的孔中。4℃孵育过夜，用DPBS洗四次，每次3 min。用1%的BSA 37℃封闭2h，DPBS洗三次，每次3 min。将5’端生物素修饰的wh3核酸适体和DNA-Lib 95℃变性10 min，置于冰上10 min；然后分别取20nM加入包被了ANXA2蛋白的酶标板孔中，室温孵育2 h。DPBS洗三次，每次3 min。在酶标板孔中，加入200μL的辣根过氧化物酶标记链霉亲和素（碧云天，P0013，A0303），室温孵育1 h。DPBS洗三次，每次3 min。在酶标板孔中，加入100μL显色液（EL-ABTS 显色试剂盒，C510031，上海生工），显色15 min。酶标仪405nm/650nm双波长检测吸光度。
结果显示（参见图3）：通过wh3核酸适体与ANXA2结合的亲和力分析，结果表明wh3核酸适体能与ANXA2特异结合。
实施例4
将对数生长期的MM.1S和RPMI-8226细胞2×105个/mL铺至24孔板中。生长过夜后，第二天加入1μg/mL的ANXA2蛋白；同时分别加入4μM的DNA-lib和wh3核酸适体。继续培养72h，细胞计数，观察ANXA2对细胞生长的刺激作用和wh3核酸适体对细胞生长的阻断情况。
结果显示（参见图4）：ANXA2蛋白能明显促进MM.1S和RPMI-8226细胞的生长，DNA-lib对ANXA2蛋白促进MM.1S和RPMI-8226细胞的生长无影响，而wh3核酸适体阻断ANXA2蛋白促进MM.1S和RPMI-8226细胞的生长。
实施例5
将HS-5细胞1×105个/mL铺至24孔板中。生长过夜后，第二天加入加入1×106个MM.1S和RPMI-8226细胞；并分别加入4μM的DNA-lib和wh3核酸适体，继续培养72 h。然后将上层培养的悬浮细胞，吸到96孔板中，用CCK-8试剂盒检测细胞活性。
结果显示（参见图5）：ANXA2核酸适体能阻断HS-5细胞促进MM.1S和RPMI-8226细胞的增殖。
实施例 6
ANXA2重组蛋白用结合缓冲液稀释到1 μg/mL，取200 μL加入到96孔板中。4℃孵育过夜，用DPBS洗四次，每次3 min。用1%BSA 37℃封闭2 h，DPBS洗三次，每次3 min。将MM.1S细胞和RPMI-8226细胞用DiI（碧云天，C1036）染色20 min。取100 μL加入已经孵育ANXA2蛋白的96孔板的孔中，同时分别加入4 μM的DNA-lib和wh3核酸适体，与细胞共同孵育2 h。用DPBS洗3次，每次2 min。通过全波长酶标仪检测荧光（激发光549 nm，发射光635 nm）。
附图6结果显示，wh3核酸适体抑制MM.1S和RPMI-8226细胞对ANXA2蛋白的粘附作用。
<110> 中南大学，湖南大学
<120> 一种特异结合膜联蛋白A2的核酸适体wh3及用途
<160> 3
<210> 1
<211> 19
<212> DNA
<400> 1
ACC GACCGT GCT GGA CTC A 19
<210> 2
<211> 19
<212> DNA
<400> 2
CGC CAG GCT CGC TCA TAG T 19
<210> 3
<211> 80
<212> DNA
<400> 3
ACCGACCGTGCTGGACTCAGGCCGATTGACTTTCCAACAAACTTAGGCCCACTAGACAGAAACTATGAGCGAGCCTGGCG 80
Claims (6)
1.特异结合膜联蛋白A2的核酸适体wh3，其特征在于：所述核酸适体wh3序列如SEQ IDNO.3所示。
2.根据权利要求1 所述特异结合膜联蛋白A2的核酸适体wh3，其特征在于：所述核酸适体wh3被用生物素进行标记。
3.如权利要求1-2任一项所述特异结合膜联蛋白A2的核酸适体wh3在制备检测膜联蛋白A2的试剂中的应用。
4.权利要求1-2任一项所述特异结合膜联蛋白A2的核酸适体wh3在纯化和浓缩膜联蛋白A2中的应用。
5.如权利要求4所述的应用，其特征在于：膜联蛋白A2蛋白与生物素标记的所述核酸适体wh3结合，然后加入链霉亲和素磁珠孵育，通过磁力板将膜联蛋白A2纯化和浓缩。
6.如权利要求1-2任一项所述特异结合膜联蛋白A2的核酸适体wh3在制备靶向膜联蛋白A2药物中的应用。
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