CN104745590A - 可检测癌症病人血清内enox2蛋白的核酸适配子及其应用 - Google Patents
可检测癌症病人血清内enox2蛋白的核酸适配子及其应用 Download PDFInfo
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Abstract
本发明实施例提供了一种可检测癌症病人血清内ENOX2蛋白的核酸适配子，其核苷酸序列如SEQ ID NO.1、SEQ ID NO.2、SEQ ID NO.3和SEQ ID NO.4所示，本发明核酸适配子的分辨率比抗体高，有非常高的特异性，用ENOX2肽链筛选得到的核酸适配子只结合ENOX2蛋白，不与其他蛋白相结合。此外，本发明核酸适配子具有非常高的亲和力：以蛋白质为靶物质，筛得适配子的解离常数可以达到nmo/L水平，甚至pmol/L水平；且一旦适配子的核酸序列确定，生产成本非常低，而且每次合成生产出的核酸适配子保持绝对一致。
Description
技术领域
本发明属于生物医学检测分析技术领域，具体涉及一种可检测癌症病人血清内ENOX2蛋白的核酸适配子及其应用。
背景技术
烟酰胺腺嘌呤二核苷酸氧化酶二硫键交换蛋白2(Ecto-Nicotinamide Adenine DinucleotideOxidase Disulfide-Thiol Exchanger 2(ENOX2))是调节细胞生长和分裂的膜蛋白。恶性肿瘤细胞在分裂和增殖过程中，ENOX2蛋白大量表达并被切割进入血液中，而非癌症病人的血清中则不存在ENOX2蛋白。在癌症发生的早期，血清中即出现ENOX2蛋白。因此，检测血液中的ENOX2蛋白可以进行癌症的早期诊断。在不同的肿瘤细胞中，ENOX2mRNA进行选择性剪接，产生选择性剪接变体ENOX2。不同种类的癌症病人血清中，可检测出不同的选择性剪接变体，其分子量和等电点不同。所以，只需要检测病人血清中的ENOX2蛋白，根据其分子量和等电点即可知道病人患有哪种癌症。
ENOX2的单链重组抗体(ScFv)可用于癌症的早期诊断，虽然ENOX2单链重组抗体不能直接识别不同癌症细胞的ENOX2选择性剪接变体，但是，利用双向电泳可以将不同分子量和等电点的ENOX2蛋白进行分离。分离后转移到膜上即可用ENOX2的重组抗体进行检测。检测癌症病人血清中的ENOX2蛋白的等电点和分子量，从而确定病人患有哪种癌症。同时，ENOX2蛋白也可作为药物靶点，通过抑制癌细胞的ENOX2蛋白的表达或活性，则有望开发出有效的治疗癌症的药物。
而现有技术是用抗ENOX2蛋白的单链重组抗体结合此蛋白，并对血清中的ENOX2进行检测。
现有技术是利用分子生物学方法，克隆获得ENOX2重组单链抗体ScFv并将其植入大肠杆菌表达质粒pET-11a。ScFv在大肠杆菌里通过IPTG诱导表达，产生大量的ENOX2重组单链抗体。大肠杆菌里所表达的抗体是没有活性的，然后通过不同的生物化学技术，包括透析法和稀释法等，对抗体进行变性和复性处理，获得有活性的ENOX2重组单链抗体。
为了检测大肠杆菌表达的ENOX2重组单链抗体的活性，现有技术克隆了人类ENOX2基因，并在大肠杆菌中进行大量表达。然后，我们把表达的ENOX2蛋白从大肠杆菌中进行提取和纯化，用于检测ENOX2重组单链抗体的活性。首先，用SDS-PAGE电泳将ENOX2蛋白分离到PAGE胶内，然后转移到硝化纤维膜上，再用ENOX2重组单链抗体作为第一抗体，抗S-tag-AP作为第二抗体，对硝化纤维膜上的ENOX2进行检测。
现有技术用抗S-tag-AP抗体结合到ENOX2重组单链抗体的S-tag上，然后用AP的底物进行染色。如果病人血清里含有ENOX2蛋白，加入AP底物后，在膜上就能显示蓝色的斑点。检测结果表明，ENOX2重组单链抗体具有结合蛋白的活性，能够在膜上检测出癌症病人血清中的ENOX2蛋白，在正常人的血清中则检测不到ENOX2蛋白的存在。
虽然现有技术通过ENOX2重组抗体也能够检测到ENOX2蛋白，但这一技术存在诸多缺点和不足：(1)在很多情况下，无论单克隆抗体、多克隆抗体或者重组单链抗体，除了结合其特异性抗原外，也对其他蛋白等有结合作用，因此特异性不够强；(2)重组单链抗体的亲和力不高；(3)重组单链抗体的生产过程复杂，生产成本高，而且在大肠杆菌里表达后，必须对重组抗体进行变性和复性。因此，不同的生产批次极易发生变化，使得重组抗体的特异性、亲和力及分辨率等发生变化。
发明内容
解决的技术问题：
针对现有技术中的上述缺陷和问题，本发明的目的在于克服现有技术的不足提供一种可检测癌症病人血清内ENOX2蛋白的核酸适配子及其应用，本发明针对ENOX2蛋白的一个序列进行核酸适配子的筛选，筛选出的适配子既可以用于ENOX2的检测，从而进行各种癌症的早期诊断，又能针对ENOX2蛋白进行抑制，从而研究开发治疗癌症的新药，本发明克服了重组单链抗体的许多缺点，获得具有对ENOX2蛋白特异性、分辨率以及亲和力高的核酸适配子。
技术方案：
为了达到上述目的，本发明实施例提供如下技术方案：
可检测癌症病人血清内ENOX2蛋白的核酸适配子，其核苷酸序列如SEQ ID NO.1、SEQID NO.2、SEQ ID NO.3和或SEQ ID NO.4所示：
SEQ ID NO.1：GGATAAGCCG CATTAGCTTA CTTCAGTGAT GCTTAAGATC TAGTA；
SEQ ID NO.2：GCTTACGCGG CATTCGCTTA CTTCACTGTA GCTTCAGTTC TGCTA；
SEQ ID NO.3：GCAGTAGCCG CTTTCGCTTA GTTCATCCTA CTTTCAGATC TACTA；
SEQ ID NO.4：GGAGTACGGC TTATTCCTAT TGATACTGCA CTTACTGGTG TAGCC。
所述的可检测癌症病人血清内ENOX2蛋白的核酸适配子，其中，所述ENOX2蛋白的保守序列如SEQ ID NO.5所示：
SEQ ID NO.5：EEAKEK FKQALSGILI QFE
即
Glu Glu Ala Lys Glu Lys Phe Lys Gln Ala Leu Ser Gly Ile Leu Ile Gln Phe Glu。
1 5 10 15
所述的ENOX2蛋白的保守序列合成的ENOX2肽链，其氨基酸序列如SEQ ID NO.6所示：
SEQ ID NO.6：EEAKEKFKQALSGILIQFE
即
Ac Glu Glu Ala Lys Glu Lys Phe Lys Gln Ala Leu Ser Gly Ile Leu Ile Gln Phe Glu Amide。
1 5 10 15
用于筛选上述可检测癌症病人血清内ENOX2蛋白的核酸适配子的DNA库，其核苷酸序列如SEQ ID NO.7所示，其中，N为ATGC中的任何一个，共45个N：
SEQ ID NO.7：
GGT ATT GAG GGT CGC ATC NNN NNN NNN NNN NNN NNN NNN NNN NNN NNN NNNNNN NNN NNN NNN NNN GAT GGC TCT AAC TCT CCT CT。
作为上述技术方案的优选，所述的可检测癌症病人血清内ENOX2蛋白的核酸适配子应用于在癌症早期诊断及通过抑制ENOX2蛋白的活性。
核酸适配子(Aptamer)是人工合成的核酸序列，能以非常高的亲合力同各种靶分子(小分子、蛋白质，甚至整个细胞)特异性结合。其功能类似抗体，但比抗体具有更多的优势。核酸适配子是通过SELEX技术，即指数富集的配基系统进化技术(SystematicEvolution of Ligandsby Exponential Enrichment，SELEX)筛选出来的。利用该技术可以从随机单链核酸序列库中筛选出特异性与靶物质高度亲和的核酸适配子。
SELEX技术的基本思想是体外化学合成一个单链寡核苷酸库，用它与靶物质混合，混合液中存在靶物质与核酸的复合物，洗掉未与靶物质结合的核酸，分离与靶物质结合的核酸分子，以此核酸分子为模板进行PCR扩增，进行下一轮的筛选。通过重复的筛选与扩增，一些与靶物质不结合或与靶物质有低亲和力、中亲和力的DNA或RNA分子被洗去，而适配子即与靶物质有高亲和力的DNA或RNA从非常大的随机文库中分离出来，且纯度随SELEX过程的进行而增高，从P摩尔到n摩尔，最后占据库的大多数(>90％左右)。
首先，本发明合成了单链DNA随机寡核苷酸文库，文库容量大于1010个单链寡核苷序列。文库的中间为随机序列，序列长度为45个核苷酸。在随机文库中，单链随机寡核苷酸分子易形成多种三维空间立体结构，几乎能与自然界所有存在的种类分子作用。由于DNA相对RNA生产成本低，体内较稳定，不易被降解，所构建的文库筛选出的适配子更适合体外诊断和体内治疗，因此，本发明选用了DNA文库。
本发明针对ENOX2蛋白的一个保守序列进行核酸适配子的筛选。首先，将ENOX2蛋白的保守序列的多肽结合到磁珠上，然后把单链DNA库与磁珠上的E的保守序列NOX2肽链结合，去除未结合的DNA，将结合的DNA洗脱出来，用PCR方法将洗脱的DNA进行扩增。PCR产物中的一条链带有生物素(Biotin)，用链霉亲和素磁珠把带有生物素的单链去掉，产生新的单链DNA进行下一轮的筛选。筛选十五轮后，将PCR产物克隆到载体进行DNA测序，最终获得与ENOX2紧密结合的特异性适配子。
有益效果
第一，核酸适配子的分辨率比抗体高：在很多情况下，无论单克隆抗体、多克隆抗体或者重组单链抗体，除了结合其特异性抗原外，也对其他蛋白等有结合作用，因此特异性不够强。而核酸适配子则有非常高的特异性，用ENOX2肽链筛选得到的核酸适配子只结合ENOX2蛋白，不与其他蛋白相结合。
第二，核酸适配子具有非常高的亲和力：以蛋白质为靶物质，筛得适配子的解离常数可以达到nmo/L水平，甚至pmol/L水平，而重组单链抗体则不能达到如此高的亲和力。
第三，核酸适配子的实用性较强：筛选适配子可定时、定量且保质，适配子的变性和复性快速、可逆，可重复使用、长期保存和室温运输，相对抗体而言，核酸适配子则具有很强的实用性。
第四，重组单链抗体的生产过程复杂，生产成本高。而且在大肠杆菌里表达后，必须对重组抗体进行变性和复性。因此，不同的生产批次极易发生变化，使得重组抗体的特异性、亲和力及分辨率等发生变化。而本发明所产生的核酸适配子则没有这个缺陷，一旦适配子的核酸序列确定，生产成本非常低，而且每次合成生产出的核酸适配子保持绝对一致。
附图说明
为了更清楚地说明本发明实施例或现有技术中的技术方案，下面将对实施例或现有技术描述中所需要使用的附图作简单地介绍，显而易见地，下面描述中的附图仅仅是本实用新型的一些实施例，对于本领域普通技术人员来讲，在不付出创造性劳动性的前提下，还可以根据这些附图获得其他的附图。
图1为本发明实施例可检测癌症病人血清内ENOX2蛋白的核酸适配子SEQ ID NO.1(Apt-1)、SEQ ID NO.2(Apt-2)、SEQ ID NO.3(Apt-3)和或SEQ ID NO.4(Apt-4)结合ENOX2的Western blot图。
图2为本发明实施例可检测癌症病人血清内ENOX2蛋白的核酸适配子SEQ ID NO.1(Apt-1)结合ENOX2的Western blot图。
具体实施方式
下面将结合发明的附图，通过具体实施方式对本发明的技术方案进行清楚、完整地描述，显然，所描述的实施例仅仅是本发明一部分实施例，而不是全部的实施例。基于本发明中的实施例，本领域普通技术人员在没有作出创造性劳动前提下所获得的所有其他实施例，都属于本发明保护的范围。
本领域技术人员将会理解，下列实施例仅用于说明本发明，而不应视为限定本发明的范围。实施例中未注明具体技术或条件者，按照本领域内的文献所描述的技术或条件或者按照产品说明书进行。所用试剂或仪器未注明生产厂商者，均为可以通过市购获得的常规产品。
以下是人类ENOX2蛋白的氨基酸序列：
SEQ ID NO.11：Human ENOX2protein sequence(from AAB30428):
MQRDFRWLWV YEIGYAADNS RTLNVDSTAM TLPMSDPTAW ATAMNNLGMA
PLGIAGQPIL PDFDPALGMM TGIPPITPMM PGLGIVPPPI PPDMPVVKEI
IHCKSCTLFP PNPNLPPPAT RERPPGCKTV FVGGLPENGT EQIIVEVFEQ
CGEIIAIRKS KKNFCHIRFA EEYMVDKALY LSGYRIRLGS STDKKDTGRL
HVDFAQARDD LYEWECKQRM LAREERHRRR MEEERLRPPS PPPVVHYSDH
ECSIVAEKLK DDSKFSEAVQ TLLTWIERGE VNRRSANNFY SMIQSANSHV
RRLVNEKAAH EKDMEEAKEK FKQALSGILI QFEQIVAVYH SASKQKAWDH
FTKAQRKNIS VWCKQAEEIR NIHNDELMGI RREEEMEMSD DEIEEMTETK
ETEESALVSQ AEALKEENDS LRWQLDAYRN EVELLKQEQG KVHREDDPNK
EQQLKLLQQA LQGMQQHLLK VQEEYKKKEA ELEKLKDDKL QVEKMLENLK
EKESCASRLC ASNQDSEYPL EKTMNSSPIK SEREALLVGI ISTFLHVHPF
GASIEYICSY LHRLDNKICT SDVECLMGRL QHTFKQEMTG VGASLEKRWK
FCGFEGLKLT
即：
Met Gln Arg Asp Phe Arg Trp Leu Trp Val Tyr Glu Ile Gly Tyr Ala
Ala Asp Asn Ser Arg Thr Leu Asn Val Asp Ser Thr Ala Met Thr Leu
Pro Met Ser Asp Pro Thr Ala Trp Ala Thr Ala Met Asn Asn Leu Gly
Met Ala Pro Leu Gly Ile Ala Gly Gln Pro Ile Leu Pro Asp Phe Asp
Pro Ala Leu Gly Met Met Thr Gly Ile Pro Pro Ile Thr Pro Met Met
Pro Gly Leu Gly Ile Val Pro Pro Pro Ile Pro Pro Asp Met Pro Val
Val Lys Glu Ile Ile His Cys Lys Ser Cys Thr Leu Phe Pro Pro Asn
Pro Asn Leu Pro Pro Pro Ala Thr Arg Glu Arg Pro Pro Gly Cys Lys
Thr Val Phe Val Gly Gly Leu Pro Glu Asn Gly Thr Glu Gln Ile Ile
Val Glu Val Phe Glu Gln Cys Gly Glu Ile Ile Ala Ile Arg Lys Ser
Lys Lys Asn Phe Cys His Ile Arg Phe Ala Glu Glu Tyr Met Val Asp
Lys Ala Leu Tyr Leu Ser Gly Tyr Arg Ile Arg Leu Gly Ser Ser Thr
Asp Lys Lys Asp Thr Gly Arg Leu His Val Asp Phe Ala Gln Ala Arg
Asp Asp Leu Tyr Glu Trp Glu Cys Lys Gln Arg Met Leu Ala Arg Glu
Glu Arg His Arg Arg Arg Met Glu Glu Glu Arg Leu Arg Pro Pro Ser
Pro Pro Pro Val Val His Tyr Ser Asp His Glu Cys Ser Ile Val Ala
Glu Lys Leu Lys Asp Asp Ser Lys Phe Ser Glu Ala Val Gln Thr Leu
Leu Thr Trp Ile Glu Arg Gly Glu Val Asn Arg Arg Ser Ala Asn Asn
Phe Tyr Ser Met Ile Gln Ser Ala Asn Ser His Val Arg Arg Leu Val
Asn Glu Lys Ala Ala His Glu Lys Asp Met Glu Glu Ala Lys Glu Lys
Phe Lys Gln Ala Leu Ser Gly Ile Leu Ile Gln Phe Glu Gln Ile Val
Ala Val Tyr His Ser Ala Ser Lys Gln Lys Ala Trp Asp His Phe Thr
Lys Ala Gln Arg Lys Asn Ile Ser Val Trp Cys Lys Gln Ala Glu Glu
Ile Arg Asn Ile His Asn Asp Glu Leu Met Gly Ile Arg Arg Glu Glu
Glu Met Glu Met Ser Asp Asp Glu Ile Glu Glu Met Thr Glu Thr Lys
Glu Thr Glu Glu Ser Ala Leu Val Ser Gln Ala Glu Ala Leu Lys Glu
Glu Asn Asp Ser Leu Arg Trp Gln Leu Asp Ala Tyr Arg Asn Glu Val
Glu Leu Leu Lys Gln Glu Gln Gly Lys Val His Arg Glu Asp Asp Pro
Asn Lys Glu Gln Gln Leu Lys Leu Leu Gln Gln Ala Leu Gln Gly Met
Gln Gln His Leu Leu Lys Val Gln Glu Glu Tyr Lys Lys Lys Glu Ala
Glu Leu Glu Lys Leu Lys Asp Asp Lys Leu Gln Val Glu Lys Met Leu
Glu Asn Leu Lys Glu Lys Glu Ser Cys Ala Ser Arg Leu Cys Ala Ser
Asn Gln Asp Ser Glu Tyr Pro Leu Glu Lys Thr Met Asn Ser Ser Pro
Ile Lys Ser Glu Arg Glu Ala Leu Leu Val Gly Ile Ile Ser Thr Phe
Leu His Val His Pro Phe Gly Ala Ser Ile Glu Tyr Ile Cys Ser Tyr
Leu His Arg Leu Asp Asn Lys Ile Cys Thr Ser Asp Val Glu Cys Leu
Met Gly Arg Leu Gln His Thr Phe Lys Gln Glu Met Thr Gly Val Gly
Ala Ser Leu Glu Lys Arg Trp Lys Phe Cys Gly Phe Glu Gly Leu Lys
Leu Thr)
实施例1
1.合成出肽链(肽链由上海强耀生物科技有限公司合成)：
Ac-EEAKEKFKQALSGILIQFE-Amide：(即
Ac-GluGluAlaLysGluLysPheLysGlnAlaLeuSerGlyIleLeuIleGlnPheGlu-Amide)(SEQ ID NO.5)
2.合成用于筛选ENOX2肽链适配子的DNA库(DNA库由苏州金唯智生物科技有限公司合成)：
Apt-L:5’-GGT ATT GAG GGT CGC ATC NNN NNN NNN NNN NNN NNN NNN NNNNNN NNN NNN NNN NNN NNN NNN NNN GAT GGC TCT AAC TCT CCT CT(SEQ IDNO.6)，PAGE纯化。其中N可以是ATGC中的任何一个，共45个N。因此，这个DNA库应该有445种不同的核苷酸序列。从如此大的库里筛选出对ENOX2保守序列的肽链特异性以及高亲和性的适配子。
3.合成三个短DNA片段，用于PCR的引物：
Apt-F:5’-GGT ATT GAG GGT CGC ATC(SEQ ID NO.8)
Apt-R:5’-AGA GGA GAG TTA GAG CCA TC(SEQ ID NO.9)
Apt-RB:5’Biotin-AGA GGA GAG TTA GAG CCA TC(SEQ ID NO.10)
Apt-L,Apt-F,Apt-R和Apt-RB都溶于10mM Tris,pH 8.0,1mM EDTA(TE缓冲液)，最终浓度100微摩。
4.将多肽结合到磁珠上，具体方法为：
(1)称量1毫克Dynabeads M-270Epoxy(Life Technologies)，放入2毫升的试管里；
(2)往试管里加入1毫升Life Technologies公司的C1溶液，混匀；
(3)将试管置入磁珠架上1分钟，使磁珠吸附到试管壁上，吸去上清液；
(4)往试管里加入20微克的多肽(15微升的1毫克/100微升溶液)，再加入235微升的C1溶液；
(5)往试管里加入250微升的Life Technologies公司的C2溶液；
(6)在37℃摇床内保温24小时，摇床保持每分钟200转，以保证磁珠始终不贴到试管壁上；
(7)24小时后，将试管置入磁珠架上1分钟，使磁珠吸附到试管壁上，吸去上清液；
(8)HB溶液洗脱：往试管里加入800微升Life Technologies公司的HB(加入tween-20至最终浓度0.05％)溶液，混匀；
(9)将试管置入磁珠架上1分钟，使磁珠吸附到试管壁上，吸去上清液；
(10)LB溶液洗脱：往试管里加入800微升Life Technologies公司的LB(加入tween-20至最终浓度0.05％)溶液，混匀；
(11)将试管置入磁珠架上1分钟，使磁珠吸附到试管壁上，吸去上清液；
(12)SB溶液洗脱：往试管里加入800微升Life Technologies公司的SB溶液，混匀。
(13)将试管置入磁珠架上1分钟，使磁珠吸附到试管壁上，吸去上清液；
(14)重复SB溶液洗脱一次；
(15)SB溶液长洗脱：往试管里加入800微升Life Technologies公司的SB溶液，混匀。室温摇15分钟；
(16)将试管置入磁珠架上1分钟，使磁珠吸附到试管壁上，吸去上清液；
(17)往试管里加入500微升的SB，混匀。置入4℃冰箱待用。
5.筛选针对ENOX2蛋白多肽的核酸适配子
(1)往1.5毫升试管里加入50微升的PBST，再加入10微升的100微摩尔浓度的ssDNA库，95℃2分钟，置入冰上10分钟；
(2)将上面热处理过的ssDNA库加入到48毫升的PBST，加入50微升1mg/ml BSA，5微升1mg/ml poly dI:dC，50微升多肽-磁珠；
(3)将48毫升的ssDNA库分装入两个50毫升的试管，放入摇床在室温摇动保温30分钟；
(4)30分钟后，将48毫升的ssDNA库装入24个2毫升的试管，放到磁珠架上，移去上清液；
(5)用500微升PBST将24个试管里的磁珠洗下，置入一个2毫升的试管里；
(6)用PBST洗磁珠两次；
(7)移去上清后，加入138微升的水与磁珠混合；
(8)将138微升的混合液分装到6个PCR管，每个管子23微升；
(9)在每个试管加入1微升Apt-F和1微升Apt-FR(100□□M),最终浓度为2□M，25微升Lucigen Taq98,Hot Start 2X Master Mix；
(10)PCR反应：95℃,4分钟；95℃30秒，56℃30秒，68℃30秒，重复30次；68℃4分钟；
(11)PCR反应后，将6个试管的PCR产物移入一个试管，将试管放到磁珠架上，上清移入到干净试管里(PCR(1))；
(12)用10微升PCR(1)产物跑2％琼脂糖凝胶电泳，加入GeneGreen，1X TAE缓冲液，100V，45分钟；
(13)UV灯观察PCR(1)产物，在85bp左右处有一条明显条带；
(14)在250微升的PCR(1)产物中，加入64微升的5M氯化钠，2微克M-280Streptavidin磁珠(使用前用PBST洗两次，每次1毫升)，摇床室温孵育10分钟；
(15)用PBST洗磁珠，每次1毫升，共洗3次；
(16)往带有磁珠的试管中加入140微升新鲜配制的100mM的氢氧化钠，摇动室温孵育5分钟；
(17)将试管放到磁珠架上，ssDNA移入到干净试管里；
(18)往试管里加入1毫升PBST，10微升的100mM磷酸缓冲液，pH 7.5；
(19)将试管在95℃加热4分钟，然后立即放入冰上，10分钟；
(20)将变性的ssDNA加入到50毫升的PBST(含有50微升的1mg/ml BSA,5微升1mg/ml的poly dI；dC，以及20微升的多肽-磁珠)，然后分成两个50毫升的试管里；
(21)将两个试管放入摇床，摇动室温孵育30分钟；
(22)在磁珠架上放入2毫升试管，将50毫升的ssDNA/多肽-磁珠溶液分装到25个2毫升试管里；
(23)移去上清液，将25个试管里的磁珠合并到一个2毫升试管里；
(24)用1毫升PBST洗磁珠两次；
(25)用93微升的去离子水将磁珠混匀，分装到4个PCR试管里，每管23微升，然后加入1微升Apt-F，1微升Apt-RB,25微升Lucigen Taq98,Hot Start 2X Master Mix；
(26)PCR反应：PCR反应：95℃,4分钟；95℃30秒，56℃30秒，68℃30秒，重复25次；68℃4分钟；
(27)将4管PCR产物合并到1个试管里，PCR产物放到磁珠架上，上清移入到干净试管里(PCR(2))；
(28)用10微升PCR(2)产物跑2％琼脂糖凝胶电泳，加入GeneGreen，1X TAE缓冲液，100V，45分钟；
(29)UV灯观察PCR(2)产物，在85bp左右处有一条明显条带；
(30)在150微升的PCR(2)产物里，加入38微升的5M氯化钠，1毫克的M-280Streptavidin磁珠(使用前用1毫升的PBST洗两次)，摇动室温孵育10分钟；
(31)用1毫升PBST洗三次；
(32)在试管里加入50微升新配制的100mM的氢氧化钠，室温5分钟；
(33)将试管放到磁珠架上，ssDNA移入干净试管里；
(34)往试管里加入1毫升PBST，10微升的100mM磷酸缓冲液pH 7.5；
(35)将试管加热95℃，4分钟，立即放入4℃水中，10分钟；
(36)将变性的ssDNA加入到50毫升的PBST(含有50微升的1mg/ml BSA,5微升1mg/ml的poly dI；dC，以及20微升的多肽-磁珠)，然后分装到两个50毫升的试管里；
(37)将两个试管放入摇床，摇动室温孵育30分钟；
(38)在磁珠架上放入2毫升试管，将50毫升的ssDNA/多肽-磁珠溶液分装到25个2毫升试管里；
(39)移去上清液，将25个试管里的磁珠合并到一个2毫升试管里；
(40)用1毫升PBST洗磁珠两次；
(41)用93微升的去离子水将磁珠混匀，分装到4个PCR试管里，每管23微升，然后加入1微升Apt-F，1微升Apt-RB,25微升Lucigen Taq98,Hot Start 2X Master Mix；
(42)PCR反应：PCR反应：95℃,4分钟；95℃30秒，56℃30秒，68℃30秒，重复20次；68℃4分钟；
(43)将4管PCR产物合并到1个试管里，PCR产物放到磁珠架上，上清移入到干净试管里(PCR(3))；
(44)用10微升PCR(3)产物跑2％琼脂糖凝胶电泳，加入GeneGreen，1X TAE缓冲液，100V，45分钟；
(45)UV灯观察PCR(3)产物，在85bp左右处有一条明显条带；
(46)反向筛选磁珠：往试管里加入50微升的无多肽的磁珠M-270，用1毫升的PBST洗两次；
(47)往150微升的PCR(3)加入38微升的5M氯化钠，1微克M-280-Streptavidin磁珠(使用前用1毫升PBST洗两次)；
(48)室温摇动孵育10分钟；
(49)用1毫升PBST洗三次；
(50)往试管里加入50微升的新鲜配制的100mM氢氧化钠，室温孵育5分钟；
(51)将试管放到磁珠架上，ssDNA上清液移入干净试管里；
(52)往ssDNA试管里加入1毫升PBST，10微升的100mM磷酸缓冲液，pH 7.5；
(53)将ssDNA试管加热95℃，4分钟，立即放入4℃水中；
(54)将ssDNA加入到M-270磁珠试管里(46)，室温孵育10分钟；
(55)将试管放到磁珠架上，反向筛选后的ssDNA移入到干净试管里(ssDNA C1)；
(56)往含有M-270的磁珠试管里加入93微升的水，混匀；
(57)PCR反应：23微升含有磁珠的水，1微升Apt-F，1微升Apt-RB,25微升LucigenTaq98,Hot Start 2X Master Mix。95℃,4分钟；95℃30秒，56℃30秒，68℃30秒，重复20次；68℃4分钟；
(58)用10微升跑2％琼脂糖凝胶电泳，加入GeneGreen，1X TAE缓冲液，100V，45分钟；
(59)UV灯观察PCR产物，在85bp左右处有一条明显条带；
(60)将150微升ssDNA C1加热95℃,4分钟，立即放入4℃水里；
(61)将变性的ssDNA C1加入到50毫升的PBST(含有50微升的1mg/ml BSA,5微升1mg/ml的poly dI；dC，以及20微升的多肽-磁珠)，然后分装到两个50毫升的试管里；
(62)将两个试管放入摇床，摇动室温孵育30分钟；
(63)在磁珠架上放入2毫升试管，将50毫升的ssDNA/多肽-磁珠溶液分装到25个2毫升试管里；
(64)移去上清液，将25个试管里的磁珠合并到一个2毫升试管里；
(65)用1毫升PBST洗磁珠两次；
(66)用93微升的去离子水将磁珠混匀，分装到4个PCR试管里，每管23微升，然后加入1微升Apt-F，1微升Apt-RB,25微升Lucigen Taq98,Hot Start 2X Master Mix；
(67)PCR反应：95℃,4分钟；95℃30秒，56℃30秒，68℃30秒，重复15次；68℃4分钟；
(68)将4管PCR产物合并到1个试管里，PCR产物放到磁珠架上，上清移入到干净试管里(PCR(4))；
(69)用10微升PCR(4)产物跑2％琼脂糖凝胶电泳，加入GeneGreen，1X TAE缓冲液，100V，45分钟；
(70)UV灯观察PCR(4)产物，在85bp左右处有一条明显条带；
(71)后面的筛选与上述方法相同，PCR都是重复20次。其中PCR(6)，PCR(9)和PCR(12)后进行反向筛选；
(72)PCR(16)的引物用Apt-F和Apt-R；
(73)PCR(16)产物克隆到TOPO TA质粒，然后转入One Shot Top 10E.coli，提取质粒进行DNA序列测定。
测序结果(1)：
总共对40个样本进行DNA序列测定，其中18个具有相同的序列(Apt-1)，12个具有相同的序列(Apt-2)，8个具有相同的序列(Apt-3)，另外两个具有相同的序列(Apt-4)，如下：
Apt-1:GGA TAA GCC GCA TTA GCT TAC TTC AGT GAT GCT TAA GAT CTA GTA(SEQID NO.1)
Apt-2:GCT TAC GCG GCA TTC GCT TAC TTC ACT GTA GCT TCA GTT CTG CTA(SEQ IDNO.2)
Apt-3:GCA GTA GCC GCT TTC GCT TAG TTC ATC CTA CTT TCA GAT CTA CTA(SEQ IDNO.3)
Apt-4:GGA GTA CGG CTT ATT CCT ATT GAT ACT GCA CTT ACT GGT GTA GCC(SEQID NO.4)
实施例2
如图1所示，为本发明实施例可检测癌症病人血清内ENOX2蛋白的核酸适配子SEQ IDNO.1(Apt-1)、SEQ ID NO.2(Apt-2)、SEQ ID NO.3(Apt-3)和或SEQ ID NO.4(Apt-4)结合ENOX2的Western blot图；人类ENOX2的cDNA克隆到pET-11a表达质粒里，转入大肠杆菌，用IPGE诱导表达ENOX2蛋白，然后提取包涵体内的总蛋白，测定蛋白质的浓度。取1微克包涵体总蛋白将蛋白用SDS-PAGE胶根据分子量进行分离，再将蛋白转到硝化纤维膜上进行WESTERN BLOT分析。Apt-1,Apt-2,Apt-3和Apt-4用生物素标记，生物素标记的四个核酸适配子与硝化纤维膜在室温孵化一个小时，洗脱未结合的适配子后，再用链霉亲和素-碱性磷酸酶与膜进行孵育，室温一小时后洗脱，加入碱性磷酸酶的底物进行显色反应。
如图2所示，为本发明实施例可检测癌症病人血清内ENOX2蛋白的核酸适配子SEQ IDNO.1(Apt-1)结合ENOX2的Western blot图；人类ENOX2的cDNA克隆到pET-11a表达质粒里，转入大肠杆菌，用IPGE诱导表达ENOX2蛋白，然后提取包涵体内的总蛋白，测定蛋白质的浓度。取1微克包涵体总蛋白将蛋白用SDS-PAGE胶根据分子量进行分离，再将蛋白转到硝化纤维膜上进行WESTERN BLOT分析。Apt-1用生物素标记，生物素标记的这个核酸适配子与硝化纤维膜在室温孵化一个小时，洗脱未结合的适配子后，再用链霉亲和素-碱性磷酸酶与膜进行孵育，室温一小时后洗脱，加入碱性磷酸酶的底物进行显色反应。
核酸适配子结合蛋白的功能测定，方法如下：
(1)将人类人类ENOX2蛋白的cDNA克隆到大肠杆菌表达质粒pET-11a，并将质粒转入大肠杆菌里；
(2)用IPTG处理大肠杆菌，诱导表达ENOX2蛋白；
(3)从大肠杆菌包涵体内提取ENOX2蛋白；
(4)通过10％SDS聚丙烯酰胺凝胶电泳将ENOX2蛋白分离，并通过电转将蛋白转移到硝化纤维膜上；
(5)用5％的去脂奶粉封闭硝化纤维膜，室温1小时；
(6)用生物素(Biotin)标记的Apt-1孵育硝化纤维膜，室温2小时；
(7)用洗脱缓冲液(10mM Tris,pH 7.5,150mM NaCl,0.05％Triton-100)洗膜四次，每次10分钟；
(8)用链霉亲和素-碱性磷酸酶(Streptavidin-AP)孵育硝化纤维膜，室温一小时；
(9)用洗脱缓冲液洗膜四次，每次10分钟；
(10)用AP显色液(每毫升AP缓冲液加入10微升BCIP溶液和10微升NBT溶液)显色3-30分钟。
实验结果见本发明实施例所示图1和图2。
实施例3Apt-1对ENOX2蛋白的活性抑制作用
人类ENOX2蛋白的氨基酸序列为SEQ ID NO.11.
为测定Apt-1是否能够抑制人类ENOX2的活性，本发明采用了ENOX2的还原酶活性对硫氧化还原蛋白的作用。为测定硫醇的产生，胰岛素和DTT混合后所发生的浊度反应。本发明的结果表明，加入S-tag的重组人类ENOX2蛋白后，浊度明显增强，表明有大量硫醇产生。在反应中加入Apt-1之后，明显抑制了重组人类ENOX2的还原酶活性。结果如表2所示，可见，Apt-1能够有效结合人类ENOX2蛋白，这一特异性的核酸适配子可用于检测血清中的ENOX2蛋白，进行癌症的早期诊断。同时，Apt-1结合ENOX2则能够有效抑制其活性，可用于癌症的治疗。
在本发明中，核酸适配子可以是单链RNA，用单链RNA库对ENOX2的肽链进行筛选，筛选出的单链RNA可以代替本发明的单链DNA核酸适配子进行ENOX2蛋白的检测；
核酸适配子还可以是双链DNA，用双链DNA库对ENOX2的肽链进行筛选，筛选出的双链DNA可以代替本发明的单链DNA核酸适配子进行ENOX2蛋白的检测；
核酸适配子还可以是双链RNA，用双链RNA库对ENOX2的肽链进行筛选，筛选出的双链RNA可以代替本发明的单链DNA核酸适配子进行ENOX2蛋白的检测。
表1 序列表
表1及SEQ ID NO.11列出了核酸适配子库，PCR引物、四个核酸适配子的DNA序列、ENOX2保守序列的氨基酸序列以及人类ENOX2蛋白的氨基酸序列。
表2 Apt-1抑制ENOX2的还原酶活性
| 1 | 2 | 3 | 4 | |
| 胰岛素+DTT | 100 | 100 | 100 | 100 |
| 胰岛素+DTT+ENOX2 | 378.21 | 377.98 | 365.84 | 346.92 |
| 胰岛素+DTT+ENOX2+Apt-1 | 139.22 | 116.82 | 132.85 | 129.78 |
由表2可见，重组ENOX2蛋白增加了胰岛素+DTT后的浊度，表明ENOX2蛋白具有还原酶的活性。反应中加入核酸适配子Apt-1后，抑制了ENOX2蛋白的还原酶活性，使得反应后的浊度降低。
Claims (5)
1.一种可检测癌症病人血清内ENOX2蛋白的核酸适配子，其特征在于，其核苷酸序列如
SEQ ID NO.1、SEQ ID NO.2、SEQ ID NO.3和SEQ ID NO.4所示：
SEQ ID NO.1：GGATAAGCCG CATTAGCTTA CTTCAGTGAT GCTTAAGATC TAGTA；
SEQ ID NO.2：GCTTACGCGG CATTCGCTTA CTTCACTGTA GCTTCAGTTC TGCTA；
SEQ ID NO.3：GCAGTAGCCG CTTTCGCTTA GTTCATCCTA CTTTCAGATC TACTA；
SEQ ID NO.4：GGAGTACGGC TTATTCCTAT TGATACTGCA CTTACTGGTG TAGCC。
2.根据权利要求1所述的可检测癌症病人血清内ENOX2蛋白的核酸适配子，其特征在于，所述ENOX2蛋白的保守序列如SEQ ID NO.5所示：
SEQ ID NO.5：EEAKEK FKQALSGILI QFE
即
Glu Glu Ala Lys Glu Lys Phe Lys Gln Ala Leu Ser Gly Ile Leu Ile Gln Phe Glu。
1 5 10 15
3.根据权利要求2所述的ENOX2蛋白的保守序列合成的ENOX2肽链，其特征在于，其氨基酸序列如SEQ ID NO.6所示：
SEQ ID NO.6：EEAKEKFKQALSGILIQFE
即
Ac Glu Glu Ala Lys Glu Lys Phe Lys Gln Ala Leu Ser Gly Ile Leu Ile Gln Phe Glu Amide。
1 5 10 15
4.根据权利要求1所述的可检测癌症病人血清内ENOX2蛋白的核酸适配子的DNA库，其特征在于，其核苷酸序列如SEQ ID NO.7所示，其中，N为ATGC中的任何一个，共45个N：
SEQ ID NO.7：
GGT ATT GAG GGT CGC ATC NNN NNN NNN NNN NNN NNN NNN NNN NNN NNN NNNNNN NNN NNN NNN NNN GAT GGC TCT AAC TCT CCT CT。
5.根据权利要求1所述的可检测癌症病人血清内ENOX2蛋白的核酸适配子，其特征在于，应用在癌症早期诊断及通过抑制ENOX2蛋白的活性在癌症治疗中。
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