CN106906219A - 特异结合膜联蛋白A2的核酸适体wh6及其应用 - Google Patents
特异结合膜联蛋白A2的核酸适体wh6及其应用 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wh6及其应用,wh6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,可作为ANXA2定量或定性检测试剂。进一步研究表明wh6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。wh6还可以与临床治疗MM药物结合通过作用ANXA2蛋白进行靶向递送和体内成像,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ANXA2的核酸适体,还涉及该适体的应用。
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、神经细胞和一些肿瘤细胞;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在肿瘤中的异常表达与肿瘤的发生、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的异常表达,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。并且抗体试剂稳定性差,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,本发明的目的在于提供特异结合并作用ANXA2的核酸适体。
本发明人通过采用消减筛选的策略,利用GST-ANXA2融合蛋白作为筛选靶标,获得与融合蛋白结合的DNA序列,进一步通过将GST蛋白作为负性筛选,将与GST蛋白结合的DNA序列去掉,获得与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wh6)。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的核酸适体在制备检测ANXA2试剂中的应用。通过将ANXA2蛋白与酶标板结合,然后利用生物素标记的wh6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。
本发明提供所述特异结合ANXA2的核酸适体在纯化和浓缩ANXA2中的应用。ANXA2蛋白与生物素标记的wh6核酸适体结合,然后加入链霉亲和素磁珠孵育,通过磁珠可将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。细胞膜表面的ANXA2蛋白能与多种蛋白结合发挥其生理作用,wh6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wh6阻断ANXA2蛋白促进MM.1S和RMPI-8226细胞的生长,wh6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. wh6核酸适体的二级结构;
图2.采用Western blot方法检测不同多发性骨髓瘤细胞株ANXA2蛋白的表达情况;
图3.采用流式细胞仪检测低表达ANXA2和高表达ANXA2多发性骨髓瘤细胞株与wh6的结合;
图4.Western blot检测shRNA敲低ANBL-6细胞中ANXA2的表达;
图5.流式细胞仪检测敲低ANXA2后ANBL-6细胞与wh6的结合;
图6.Aptamer-pull down实验检测wh6与ANXA2蛋白结合;
图7. wh6抑制ANXA2刺激MM.1S和RPMI-8226细胞的生长;
图8. wh6抑制HS-5细胞刺激MM.1S和RPMI-8226细胞的生长;
图9. wh6抑制MM.1S和RPMI-8226细胞与ANXA2蛋白的粘附;
图10. 体内观测荷瘤小鼠尾静脉注射Cy5-wh6和Cy5-DNA-lib文库的活体成像图;
图11. 荷瘤小鼠尾静脉注射Cy5-wh6和Cy5-DNA-lib文库的器官荧光成像图。
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,可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,库的序列为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5000 pmol,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10min。混匀包被了GST蛋白的磁珠溶液,取100 μL置于BSA包被的EP管中,用150 μL结合缓冲液洗涤3次。ssDNA文库与包被了GST蛋白的磁珠混匀,室温反应1 h,然后取上清,去除与GST蛋白结合的ssDNA序列。混匀包被了ANXA2的磁珠溶液,取100 μL置于BSA包被的EP管中,用150 μL结合缓冲液洗涤3次。将上清ssDNA文库与包被了膜联蛋白的磁珠混匀,同时加入酵母tRNA竞争结合,室温反应1 h。置磁力架2-3 min,弃上清,用1×洗涤缓冲液洗去未结合的ssDNA,如此重复3次。置磁力架3-4 min,弃上清,管中加入200 μL去离子水,95℃加热5min,置磁力架2 min,取上清为模板,进行PCR扩增。
配置PCR体系:模板2μL,引物P80-SF(10uM)1μL,引物P80-AP(10uM)1μL,dNTP(各2.5mM)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℃总延伸5 min。扩增后将PCR产物转移置一个1.5 mL的离心管中。将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,室温摇床结合1 h, 1000 rpm,离心2 min,弃上清。用1×磁珠结合洗涤缓冲液洗涤2-3次,加入400μL的200 mM NaOH孵育5 min,使dsDNA解链,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,如此重复进行9个循环。
克隆测序及序列分析
将第9轮筛选得到的寡核苷酸适体富集库用无修饰的引物扩增,PCR产物经纯化后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wh6(另一条命名为wh3,另案予以申请)。具体序列如下:
5'-ACCGACCGTGCTGGACTCAGTCCGATCTCTCCACAGAGACAAACTTAGGACCCCTAGTCCCACTATGAGCGAGCCTGGCG -3’(SEQ ID NO.3)
然后通过mfold软件行对wh6核酸适体序列二级结构进行分析,结构如图1所示。
实施例2
使用RIPA蛋白裂解液(P0013B,Beyotime生物技术研究所)提取多发性骨髓瘤ANBL-6和NCI-H929细胞的总蛋白,用BCA试剂盒(购自promega公司)测定蛋白浓度,电泳,转膜,分别与ANXA2抗体(sc86235,Santa Cruz生物公司,稀释比为1:1000)和GAPDH抗体(稀释比为1:5000)孵育,加入相应HRP标记的二抗,孵育2 h,PBST洗膜,通过ECL反应液显影。检测不同多发性骨髓瘤细胞株ANXA2蛋白的表达情况。
结果见图2,发现ANXA2在多发性骨髓瘤ANBL-6细胞中表达较高,而在NCI-H929细胞中表达较低。
实施例3
多发性骨髓瘤ANBL-6细胞和NCI-H929细胞培养至对数生长期,计数将细胞调至1×106个/mL;各取0.3 mL细胞,用Wash Buffer洗两次;将FITC荧光基团修饰的wh6和DNA-lib,用Binding Buffer稀释到250 nM;与细胞在冰水混合物中孵育50 min,用Binding Buffer洗两次,每次2 min;用PBS将细胞重悬至400 μL,经流式细胞仪检测并分析结果。
结果见图3显示wh6核酸适体与多发性骨髓瘤ANBL-6细胞有显著结合,而与多发性骨髓瘤NCI-H929细胞无明显结合。
实施例4
ANXA2-shRNA干扰序列(TRCN0000296322, TRCN0000056147)通过293T细胞进行病毒包装,并用NIH3T3细胞检测病毒滴度。ANBL-6细胞培养至对数生长期。经计数后,将细胞调至1×106个/mL;用30×106病毒感染1×106个细胞。感染病毒48小时后,收集细胞,取其中一半用流式细胞仪检测ANBL-6细胞与wh6核酸适体的结合;另一半细胞提取其总蛋白,采用Western blot检测细胞ANXA2的表达。
结果见图4,发现干扰序列能敲低ANBL-6细胞中ANXA2的蛋白表达;附图5结果显示,在ANBL-6细胞中敲低ANXA2后与wh6核酸适体的结合明显下降。
实施例5
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30min,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50 pmol的5’端生物素修饰的wh6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。然后1000 rpm,离心2min,弃上清,加入1 mL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。然后1000 rpm,离心2min,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wh6和DNA-Lib与ANXA2蛋白的结合情况。
结果显示(参见图6),DNAl-lib及链霉亲和素琼脂糖珠不与ANXA2蛋白结合,wh6能与ANXA2蛋白结合。
实施例6
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wh6核酸适体。继续培养72 h,细胞计数,观察ANXA2对细胞生长的刺激作用和wh6核酸适体对细胞生长的阻断情况。
结果显示(参见图7):ANXA2蛋白能明显促进MM.1S和RPMI-8226细胞的生长,DNA-lib对ANXA2蛋白能促进MM.1S和RPMI-8226细胞的生长无影响,而wh6能阻断ANXA2蛋白促进MM.1S和RPMI-8226细胞的生长。
实施例7
将HS-5细胞以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4 μM的DNA-lib和wh6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8):ANXA2核酸适体能阻断HS-5细胞能促进MM.1S和RPMI-8226细胞的增殖。
实施例8
ANXA2重组蛋白用结合缓冲液稀释到1μg/mL,取200μL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100μL加入已经包被ANXA2蛋白的96孔板的孔中,同时分别加入4 μM的DNA-lib和wh6核酸适体,与细胞共同孵育2 h。用DPBS洗3次,每次2 min。通过全波长酶标仪测荧光(激发光549 nm,发射光635 nm)。
结果显示(参见图9),ANXA2核酸适体抑制MM.1S.和RPMI-8226细胞对ANXA2蛋白的粘附作用。
实施例9
NCG小鼠购自南京大学动物模式研究所,雄性,4-6周大小。在小鼠的左侧背部皮下注射1×107个ARP-1细胞,肿瘤细胞生长约15-20天左右(直到肿瘤直径大小为0.5-1.5cm)。皮下成瘤的NCG小鼠通过尾静脉注射cy5-荧光标记wh6核酸适体和DNA-lib序列,在固定的时间点通过IVIS Lumina II小动物成像系统采集荧光信号。对于离体荧光实验,荷瘤小鼠静脉内注射用Cy5标记wh6或DNA-lib序列。在注射后1小时用乙醚麻醉,颈椎脱位处死。通过解剖取其脾、肺、心脏和肿瘤组织等组织,用IVIS Lumina II小动物成像系统采集荧光信号。
结果显示(参见图10):通过尾静脉注射Cy5标记wh6后,肿瘤处的荧光信号在注射后30min最强,注射60 min时荧光信号有所下降,并在注射2 h后几乎消失。而尾静脉注射Cy5标记DNA-lib的荷瘤小鼠,在肿瘤组织处无明显的荧光信号。
结果显示(参见图11):通过尾静脉注射Cy5标记wh6和DNA-lib序列1 h后,观察荧光信号的组织聚集分布结果显示,wh6主要在肿瘤组织处聚集,在肺组织,脾组织和心脏组织无荧光信号聚集。而注射DNA-lib的小鼠肿瘤组织,肺组织、脾组织和心脏组织无荧光信号的聚集。
<110> 中南大学,湖南大学
<120> 特异结合膜联蛋白A2的核酸适体wh6及其应用
<160> 3
<210> 1
<211> 19
<212> DNA
<400> 1
ACCGACCGTGCTGGACTCA 19
<210> 2
<211> 19
<212> DNA
<400> 2
CGCCAG GCTCGCTCATAGT 19
<210> 3
<211> 80
<212> DNA
<400> 3
ACCGACCGTGCTGGACTCAGTCCGATCTCTCCACAGAGACAAACTTAGGACCCCTAGTCCCACTATGAGCGAGCCTGGCG 80
Claims (8)
1.特异结合膜联蛋白A2的核酸适体wh6,其特征在于:所述核酸适体序列如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,其特征在于:所述核酸适体wh6与膜联蛋白A2特异结合。
3.权利要求1-2任一项所述特异结合膜联蛋白A2的核酸适体在制备检测膜联蛋白A2试剂中的应用。
4.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wh6检测膜联蛋白A2的方法,其特征在于:将膜联蛋白A2蛋白与酶标板结合,然后利用生物素标记的所述核酸适体wh6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膜联蛋白A2的浓度。
5.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wh6在纯化和浓缩膜联蛋白A2中的应用。
6.如权利要求5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wh6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。
7.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wh6在制备靶向膜联蛋白A2药物中的应用。
8.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wh6在ANXA2高表达的恶性肿瘤治疗中提供新的治疗方案、观察治疗效果和判断预后的应用。
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FENGMEI PI ET AL.,: "RNA Nanoparticles Harboring Annexin A2 Aptamer Can Target Ovarian Cancer for Tumor-Specific Doxorubicin Delivery", 《NANOMEDICINE》 * |
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