CN105646695A - 鳐血管生成抑制因子1的适配子k16及其筛选方法和应用 - Google Patents
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Abstract
一组能识别鳐血管生成抑制因子1功能区蛋白的适配子及其制备方法。寡核苷酸序列包括SEQ?ID?No.2~11,其分别具有较高的亲和特异性,可以用于鳐血管生成抑制因子1功能区蛋白的检测。
Description
技术领域
本发明属于生物技术领域,具体地说,本发明涉及一种鳐血管生成抑制因子1适配子及其筛选方法和应用。
背景技术
近些年来,寡核苷酸适配子作为抗体分子的前景性替代分子,其研究较为引人注目。寡核苷酸适配子是由SELEX技术(Systematicevolutionofligandsbyexponentialenrichment)生物文库技术筛选获得的,该技术的原理就是利用分子生物学技术,构建人工合成的单链随机寡核苷酸文库,其随机序列长度在20-100个碱基左右。利用单链寡核苷酸分子构像灵活多变的特性,将随机寡核苷酸文库与靶分子相互作用,保留以空间构像与靶分子结合的寡核苷酸,经反复扩增、筛选数个循环,即可使与该靶子特异结合的寡核苷酸序列得到富集,最终获得多种靶分子的特异寡核苷酸适配子,即aptamer。利用SELEX技术筛选获得的aptamer识别分子的模式与蛋白抗体类似,但与蛋白类抗体相比,核酸类配基具有更多的优越性,如不受免疫条件和免疫原性限制,可体外人工合成,变性与复性可逆,可修饰并有利于长期保存和室温运输等。更重要的是,aptamer比抗体具有更高的特异性,甚至能识别单抗不能区分的蛋白质分子。而且aptamer的靶分子非常广泛,小至染料分子,大至完整的病毒颗粒和细菌病原体,甚至完整的细胞也可以通过消减SELEX技术筛选出高亲和力的寡核苷酸适配子。
鳐血管生成抑制因子1功能区以下简称鳐功能区,鳐血管生成抑制因子1是我们首次从南海海域一种鳐组织中分离出来的蛋白质(分子量42KD)。研究结果显示:鳐血管生成抑制因子1显著抑制鸡胚绒毛尿囊膜本身的及人鼻咽癌细胞诱导的鸡胚绒毛尿囊膜血管生成;无论是腹腔注射还是灌胃都显著抑制裸小鼠Lewis肺癌的生长和转移,减少肿瘤组织微血管密度,下调促血管生成因子VEGF的表达;对裸小鼠黑色素瘤B16细胞的转移也有强抑制作用;下调促转移因子CD44v6和ErBb2的表达;与5-氟尿嘧啶(5-FU)联合应用时效果更显著。鳐血管生成抑制因子1的作用靶点与美国基因技术研究所开发出的抗癌新药Avastin不同。Avastin是基因工程产品,是VEGF的抗体,其作用靶点是VEGF;鳐血管生成抑制因子1是天然产物,它不仅作用于VEGF,还作用于bFGF和PDGF。因此,针对鳐血管生成抑制因子1功能区进行特异性的鉴别和筛选是本领域一贯的追求。
基于以上考虑,本发明以鳐血管生成抑制因子1功能区蛋白为目的靶蛋白,采用SELEX技术获得了10条鳐血管生成抑制因子1功能区蛋白特异的aptamer,组合应用能够迅速、敏感、特异的检测到鳐血管生成抑制因子1功能区蛋白。由于单链DNA寡核苷酸适配子性能稳定、合成方便且廉价、经修饰后可直接用于荧光或化学发光、发色方法检测靶绑带,因此操作简单、直接。
发明内容
本发明的目的在于提供不仅具有检测快速、操作简单、稳定性高于抗体等特点,而且制备方法容易、制备周期较短的一组能识别鳐血管生成抑制因子1功能区蛋白的寡核苷酸序列及其制备方法。
所述能识别鳐血管生成抑制因子1功能区蛋白的寡核苷酸序列,包括SEQIDNo.2-11,且采用其中的一条寡核苷酸序列就能完成对鳐血管生成抑制因子1功能区蛋白的识别检测;
所述一组能识别鳐血管生成抑制因子1功能区蛋白的寡核苷酸序列的制备方法包括以下步骤:
1、合成用于筛选的ssDNA寡核苷酸文库(5′-TCAGTCGCTTCGCCGTCTCCTTC----
N35----GCACAAGAGGGAGACCCCAGAGGG-3′),其中N35为35个随机寡核苷酸;
2、将寡核苷酸文库分别与鳐血管生成抑制因子1功能区蛋白混合后进行SELEX筛选,获得适配子富集文库;
3、SELEX筛选完成后,对获得的适配子富集文库进行克隆测序;
4、选择测序结果中出现的高拷贝ssDNA,进行亲和特异性的验证,筛选获得能识别鳐血管生成抑制因子1功能区蛋白的寡核苷酸序列。
具体实施方式
实施例1
1、鳐血管生成抑制因子1功能区蛋白的制备
采用本领域技术人员熟知的酵母重组表达的方式获得具有与鳐血管生成抑制因子1功能区蛋白相同生物活性的鳐血管生成抑制因子1功能区蛋白,该蛋白序列如SEQIDNO:1所示;蛋白溶液的浓度为15mg/ml。
2、文库和引物的合成
2.1、合成用于筛选的ssDNA寡核苷酸文库(5′-TCAGTCGCTTCGCCGTCTCCTTC----
N35----GCACAAGAGGGAGACCCCAGAGGG-3′),其中N35为35个随机寡核苷酸;
引物P1:TCAGTCGCTTCGCCGTCTCCTTC;
引物P2:CCCTCTGGGGTCTCCCTCTTGTGC。
2.2、适配子的SELEX筛选,具体方法如下:
2.2.1ssDNA与鳐血管生成抑制因子1功能区蛋白的结合、分离,具体方法如下:
取100μM的ssDNA寡核苷酸文库4μL,用2×结合缓冲液稀释至100μl,95℃变性5min,冰浴10min后加入100μl鳐血管生成抑制因子1功能区蛋白,摇床结合30min,再6000rpm离心5min,弃上清,然后用1×结合缓冲液洗沉淀,弃上清;在沉淀中再加入1×结合缓冲液100μL,96℃加热5min,然后15000rpm离心10min,取上清液,对沉淀再次加热并离心,合并上清液,则可分离得到与鳐血管生成抑制因子1功能区蛋白有亲和力的ssDNA次级文库;所述2×结合缓冲液为20×结合缓冲液用双蒸水稀释10倍后的溶液,所述1×结合缓冲液为20×结合缓冲液用双蒸水稀释20倍后的溶液;所述20×结合缓冲液配方为1MNaCl、50mMKCl、500mMTris-HCl、10mMMgCl2、pH7.4。
2.2.2ssDNA与鳐血管生成抑制因子1功能区蛋白的结合、分离,具体方法如下:
将步骤2.2.1分离得到的能与鳐血管生成抑制因子1功能区蛋白结合的ssDNA,再与100μl鳐血管生成抑制因子1功能区蛋白摇床结合30min,后续步骤同步骤2.2.1,则可分离到与鳐血管生成抑制因子1功能区蛋白都有亲和力的ssDNA次级文库。
2.2.3不对称PCR扩增ssDNA,具体方法如下:
对步骤2.2.2分离获得的ssDNA次级文库进行不对称PCR扩增,总体积为25μl的不对称PCR扩增体系为:10×PCR缓冲液:2μl;P1(10μM):1μl;P2(0.2μM):1μl;dNTP(各2.5mM):0.4μl;MgCl2(25mM):1.2μl;ssDNA模板(0.2μg/μl):2μl;TaqDNA聚合酶(5u/μl):0.2μl;ddH2O:17.2μl;PCR反应参数:94℃预变性4min,然后进行40个循环94℃变性30s,58℃退火30s,72℃延伸20s,最后72℃延伸7min;
2.2.4亲和力的测定,具体方法如下:
2.2.4.1扩增:用带有地高辛标记的引物P1不对称PCR扩增筛选出来的ssDNA次级文库,扩增条件和参数与步骤2.2.3的不对称PCR扩增体系和参数相同;
2.2.4.2与蛋白结合:取步骤2.2.4.1扩增所得的PCR产物100μL,95℃变性5min,冰浴10min后加入100μL蛋白中,充分混合,在室温下结合30min,然后6000rpm离心,分离蛋白与上清液,蛋白中包含有与蛋白中结合的带地高辛标记的ssDNA,上清液中是未结合的ssDNA,同时做一不加ssDNA的空白,即用2×结合缓冲液代替PCR产物,同样进行上述操作;
2.2.4.3洗涤:将蛋白用1×结合缓冲液500μL洗涤1次,6000rpm离心,弃上清,取蛋白;
2.2.4.4与酶标兔抗地高辛抗体结合:在蛋白中加入100μL1∶900TBS稀释的过量的酶标兔抗地高辛抗体,充分混合后,反应10min,使之与蛋白中的地高辛标记的ssDNA结合;
所述TBS为0.5MTris-NaCl溶液,配制方法为:先水溶8.5~9gNaCl,再加Tris-HCl(0.5M、pH7.6)溶液100ml,最后加水定容至1L;0.5MTris-HCl(pH7.6,100ml)溶液配制方法:称取Tris6.06g,加入双蒸水40ml溶解,滴加浓HCl调pH至7.6,定容至100ml。
2.2.4.5洗涤:6000rpm离心,去上清,再用1×结合缓冲液500μL洗涤3次,得蛋白;
2.2.4.6TMB(四甲基联苯胺)显色:加入400μL双蒸水重悬蛋白,再加入200μLTMB显色液,避光显色10min后,以2mol/LH2SO4200μL终止反应,测定450nm处的吸光值OD450,该值即反映与菌结合的ssDNA的亲和力,即OD结合,空白同样进行上述步骤2.2.4.3,2.2.4.4,2.2.4.5和2.2.4.6,得到空白相应的吸光度OD空白;
所述TMB显色液使用常规的配制方法配制。
2.2.4.7测定PCR产物中DNA的摩尔浓度:取步骤2.2.4.1扩增所得的PCR产物,以已知浓度梯度的初始ssDNA文库为标准品,用Bandscan软件作为图像分析软件,采用溴化乙锭琼脂糖凝胶电泳法定量测定PCR产物中的DNA含量,获得相应DNA的摩尔浓度,进而可以计算出100μLPCR产物中的DNA摩尔数。
2.2.4.8计算相应文库的亲和力:
2.3重复筛选,具体方法为:以每一轮不对称PCR的产物作为下一轮的筛选文库,重复上述SELEX筛选步骤2.2,直到亲和力不再上升为止,最后经过16轮的筛选得到ssDNA的适配子富集文库。经不对称PCR扩增后,条件同前面的步骤,克隆并测序,得到拷贝数最高的20个有效的ssDNA,将20个适配子分别进行亲和特异性验证,得到10个对鳐血管生成抑制因子1功能区蛋白有较好亲和特异性的寡核苷酸序列(适配子),具体序列如下:
亲和力具体数据如下:
适配子名称 | 亲和力 | 适配子名称 | 亲和力 |
K2 | 0.62 | K15 | 0.43 |
K6 | 0.53 | K16 | 0.51 |
K9 | 0.50 | K17 | 0.59 |
K10 | 0.47 | K19 | 0.62 |
K14 | 0.60 | K20 | 0.49 |
2.4、分析20条适配子的特异性和亲和性
荧光标记的适配子序列与鳐血管生成抑制因子1功能区蛋白孵育,进行流式细胞分析检测,其中10条序列显示了高荧光强度,使用GraphPadPrism5.0软件针对饱和曲线做非线性回归曲线,分别对10条高亲和性适配子序列采用相同的实验操作,得到了每条是配置的Kd值:
适配子名称 | Kd值(nM) | 适配子名称 | Kd值(nM) |
K2 | 35.27 | K15 | 59.23 |
K6 | 51.73 | K16 | 38.97 |
K9 | 43.81 | K17 | 44.83 |
K10 | 50.46 | K19 | 34.57 |
K14 | 35.89 | K20 | 47.81 |
其中K20的Kd值最小,说明与靶蛋白可以快速结合并且结构稳定不易分离。
采用DNAMAN软件构建了10条适配子的二级结构并计算了他们的最小自由能,其结构最小自由能也都较小,结构也相对稳定。
2.5适配子特异性分析
分别采用BSA、人血红蛋白、鳐血管生成抑制因子1功能区蛋白与10条适配子进行特异性检测,经过结合试验发现,这10条序列都不与BSA或人血红蛋白相结合,而只与鳐血管生成抑制因子1功能区蛋白结合保持较高的特异性。
序列表
〈110〉张勇
〈120〉鳐血管生成抑制因子1的适配子K16及其筛选方法和应用
〈160〉13
〈210〉1
〈211〉225
〈212〉PRT
〈213〉鳐
〈400〉1
TLDIYKQLRDKETPSGFTLDDVIQTGVDNPGHPFIMTVGCVAGDEESYEVFKALFDPVIQ60
DRHGGYKPTDKHKTDLNHENLKGGDDLDPNYVLSSRVRTGRSIKGIALPPHCSRGERRLV120
EKLCLEGLATLTGEFQGKYYPLTTMSDAEQQQLIDDHFLFDKPVSPLLLASGMARDWPDA180
RGIWHNNDKTFLVWVNEEDHLRVISMQKGGNMKEVFRRFCVGLKK225
〈210〉2
<211〉84
〈212〉DNA
〈213〉人工序列
〈400〉K2
1TCAGTCGCTTCGCCGTCTCCTTCATGATCGCGCTGACAAATTAGGCCATTCAATCAGAGC
61ACAAGAGGGAGACCCCAGAGGG
〈210〉3
<211〉84
〈212〉DNA
〈213〉人工序列
〈400〉K6
1TCAGTCGCTTCGCCGTCTCCTTCCCGTGATGAATTGCTGATGAGCGCAGCATGGAGCTGC
61ACAAGAGGGAGACCCCAGAGGG
〈210〉4
<211〉84
〈212〉DNA
〈213〉人工序列
〈400〉K9
1TCAGTCGCTTCGCCGTCTCCTTCTGACGCATTCGGATCCAAGTTAATTAAATAACTGCGC
61ACAAGAGGGAGACCCCAGAGGG
〈210〉5
<211〉84
〈212〉DNA
〈213〉人工序列
〈400〉K10
1TCAGTCGCTTCGCCGTCTCCTTCATTGCAACCTGAGGCCATGGGACAGACCATGATAGGC
61ACAAGAGGGAGACCCCAGAGGG
〈210〉6
<211〉84
〈212〉DNA
〈213〉人工序列
〈400〉K14
1TCAGTCGCTTCGCCGTCTCCTTCAACTTGGACCCTTGAGCGATGAAGTAACGGTTTACGC
61ACAAGAGGGAGACCCCAGAGGG
〈210〉7
<211〉84
〈212〉DNA
〈213〉人工序列
〈400〉K15
1TCAGTCGCTTCGCCGTCTCCTTCGCACTGCTACCGATATTACATATATGGAGATACAGGC
61ACAAGAGGGAGACCCCAGAGGG
〈210〉8
<211〉84
〈212〉DNA
〈213〉人工序列
〈400〉K16
1TCAGTCGCTTCGCCGTCTCCTTCGGCCGAGTAACAGATTGGAACCCAACTGAGTGAGAGC
61ACAAGAGGGAGACCCCAGAGGG
〈210〉9
<211〉84
〈212〉DNA
〈213〉人工序列
〈400〉K17
1TCAGTCGCTTCGCCGTCTCCTTCTTATGGACGAGTAGAGGTACGATGACCCAATGATTGC
61ACAAGAGGGAGACCCCAGAGGG
〈210〉10
<211〉84
〈212〉DNA
〈213〉人工序列
〈400〉K19
1TCAGTCGCTTCGCCGTCTCCTTCCGATTGAGGGAGATTACGCATATGAGTACAACTGAGC
61ACAAGAGGGAGACCCCAGAGGG
〈210〉11
<211〉84
〈212〉DNA
〈213〉人工序列
〈400〉K20
1TCAGTCGCTTCGCCGTCTCCTTCTTTAGACCCGATAATGTTGTTTTGGTGACCGAATTGC
61ACAAGAGGGAGACCCCAGAGGG
〈210〉12
<211〉23
〈212〉DNA
〈213〉人工序列
〈400〉P1
TCAGTCGCTTCGCCGTCTCCTTC
〈210〉13
<211〉24
〈212〉DNA
〈213〉人工序列
〈400〉P2
CCCTCTGGGGTCTCCCTCTTGTGC
Claims (3)
1.一种用于适配子筛选的鳐血管生成抑制因子1功能区蛋白,其序列为SEQIDNO:1所示。
2.一种识别鳐血管生成抑制因子1功能区蛋白的寡核苷酸序列,其特征在于其序列为SEQIDNo.8所示。
3.权利要求2所示的寡核苷酸序列用于筛选鳐血管生成抑制因子1功能区蛋白的应用。
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Citations (3)
Publication number | Priority date | Publication date | Assignee | Title |
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WO2006096754A2 (en) * | 2005-03-07 | 2006-09-14 | Archemix Corp. | Stabilized aptamers to psma and their use as prostate cancer therapeutics |
CN101724631A (zh) * | 2008-11-03 | 2010-06-09 | 广东海洋大学 | 鳐血管生成抑制因子1功能区的制备及在防治肿瘤药物中的应用 |
CN103060326A (zh) * | 2012-12-17 | 2013-04-24 | 集美大学 | 6条寡核苷酸序列及其应用 |
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Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO2006096754A2 (en) * | 2005-03-07 | 2006-09-14 | Archemix Corp. | Stabilized aptamers to psma and their use as prostate cancer therapeutics |
CN101724631A (zh) * | 2008-11-03 | 2010-06-09 | 广东海洋大学 | 鳐血管生成抑制因子1功能区的制备及在防治肿瘤药物中的应用 |
CN103060326A (zh) * | 2012-12-17 | 2013-04-24 | 集美大学 | 6条寡核苷酸序列及其应用 |
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