CN105911282A - 一种口腔癌特异性检测的试剂盒 - Google Patents

一种口腔癌特异性检测的试剂盒 Download PDF

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CN105911282A
CN105911282A CN201610503507.8A CN201610503507A CN105911282A CN 105911282 A CN105911282 A CN 105911282A CN 201610503507 A CN201610503507 A CN 201610503507A CN 105911282 A CN105911282 A CN 105911282A
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陈博
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Abstract

本发明公开了一种用于口腔癌检测的试剂盒。本发明提供的核酸适体,与人BPIFB2蛋白具有较好的亲和能力。利用本发明的核酸适体,可以捕获唾液中的BPIFB2蛋白,通过其含量的变化来检测口腔癌,将其制备成为相应的试剂盒,将用于口腔癌的筛查。利用本发明的试剂盒,具有高灵敏、成本低的好处。

Description

一种口腔癌特异性检测的试剂盒
本申请是申请日为2015年11月15日、申请号为201510775735.6、发明名称为“一种口腔癌特异性检测的试剂盒”的发明专利申请的分案申请。
技术领域
本发明涉及一种用于检测口腔癌的试剂盒及其检测方法。
背景技术
口腔癌(Oral Squarmous Cell Carcinomas,0SCC)是人体常见的肿瘤之一,尤其是在发展中国家印度、斯里兰卡、巴西等其发病率可以达到全部肿瘤的25%。近年来口腔癌的发病率在欧美等发达国家有明显的上升趋势,特别是发病年龄趋向年轻化。口腔鳞癌不仅发病率高,且恶性程度较高;往往造成语音、咀嚼、吞咽、面容等功能障碍和严重的面容毁损,并威胁到患者的生命;五年生存率只有60%左右。尽管近来口腔癌手术方法的不断改进和放疗、化疗的广泛应用,但口腔癌的五年生存率并没有明显的提高。导致这一状况的主要原因在于:口腔癌的发病机制不清,侵袭转移机制不明,缺乏有效的早期预防和预后判断手段。因此研究口腔癌发生和发展的分子机制,从中找到口腔癌早期预防、预后判断和治疗方法是口腔癌研究中急待解决的重大问题。
现有技术已知BPIFB2基因与口腔癌的关系:BPIFB2与口腔癌具有很好的相关性,可用于制备口腔癌辅助诊断或者预后制剂,具有重要的临床应用价值。其中BPIFB2在癌旁组织中的表达水平明显低于口腔癌瘤组织和对照质粒OOcopies,而在口腔癌组织中的BPIFB2高表达,即生物信息学筛选得到的BPIFB2基因与口腔癌具有很好的相关性,可用于制备口腔癌辅助诊断或者预后制剂。因此检测BPIFB2的表达变得尤为重要。
核酸适体(Aptamer,又称适配体,适配子)是能高亲和性、高特异性的结合某种生物革El标的单链寡核酸分子(ssDNA或ssRNA)。核酸适体是通过指数富集配体系统进化技术(Systemat1c Evolut1on of L1gands by Exponent1alenr1chment,SELEX)从人工合成的DNA/RNA文库中筛选得到的能够高度特异性结合靶标分子的单链DNA/RNA。已报道核酸适体的靶标包括金属离子、有机小分子、多肽、蛋白质、细胞甚至组织等。核酸适体的分子识别功能与抗体类似,具有与抗体分子相当甚至更强的靶标识别能力,但与抗体相比具有很多优良的特性,如分子量小,能批量生产,不易失活,无免疫原性、容易合成与标记、快速的穿透组织、良好的代谢动力学、不同批次之间产品不会存在差异和具有很好化学稳定性,在生物检测、疾病诊断治疗等领域具有重要的应用前景。
发明内容
本发明的目的是提供一种特异结合BPIFB2的核酸适体及其试剂盒。
本发明提供的核酸适体,是序列表的序列1-15所示的单链DNA。
所述核酸适体与BPIFB2蛋白具有较好的亲和能力。
还可将所述核酸适体进行修饰或改造,得到所述核酸适体的衍生物。
所述核酸适体的衍生物可为如下任意一种:
a)将所述核酸适体删除部分或增加部分互补的核苷酸,得到的与所述核酸适体具有相同功能的核酸适体的衍生物;
b)将所述核酸适体进行核苷酸取代或部分修饰,得到的与所述核酸适体具有相同功能的核酸适体的衍生物;
c)将所述核酸适体的骨架改造为硫代磷酸酯骨架,得到的与所述核酸适体具有相同功能的核酸适体的衍生物;
d)将核酸适体改造为肽核酸,得到的与所述核酸适体具有相同功能的核酸适体的衍生物;
e)将所述核酸适体连接上荧光、放射性和治疗性物质后,得到的与所述核酸适体具有相同功能的核酸适体的衍生物。
所述核酸适体可用于制备检测BPIFB2的试剂盒。
利用本发明的核酸适体,可以捕获中唾液中的中的BPIFB2,从而用于相关口腔癌筛查。利用本发明的核酸适体,具有高灵敏、成本低、易制备、易保存的优点。本发明具有很高的应用价值。
具体实施方式
以下的实施例便于更好地理解本发明,但并不限定本发明。下述实施例中的实验方法,如无特殊说明,均为常规方法。
实施例1BPIFB2蛋白的获得
将NP_079503所示的BPIFB2基因通过本领域常规的真核表达方式进行表达,获得了相应的目的多肽蛋白。
实施例2核酸适体的筛选和制备
设计两端包含大约20个核苷酸、中间包括41个核苷酸的随机核酸文库如下:
5‘-ACCTGATGCCATGCATCGCA(N41)CTAGCCAGCCTTTGACATCA-3’;N41代表41个随机核苷酸。
将单链DNA文库扩增为双链DNA,产物经2%琼脂糖凝胶电泳并切胶回收纯化;以回收的双链DNA为模板,体外转录出单链RNA随机文库,转录产物经PAGE纯化。75μg RNA文库经硝酸纤维素膜反筛去除与膜结合的RNA分子,然后与2ugBPIFB2蛋白,37℃孵育30min,反应液经硝酸纤维素膜滤过,洗涤滤膜;然后将滤膜剪碎,置于洗脱缓冲液(6mol/L尿素,0.55mol/L醋酸铵,l.5mmol/LEDTA,0.15%SDS)中煮沸5min,离心,取上清,无水乙醇沉淀RNA,并重新溶解于20μ1DEPC水中;以RNA为模板RT-PCR扩增双链DNA,体外转录出RNA文库用于下一轮筛选;每轮筛选过程中RT-PCR得到双链DNA文库,以该双链DNA为模板体外转录出RNA适配子库,筛选共进行11轮。得到了16个适配子,其序列分别为SEQ ID NO:1-16所示。具体序列如下所示:
BPIFB2-1:
ACCTGATGCCATGCATCGCATCACACGATCATCACACCGCTCCGCCTTCCAACTACCGCTCCTAGCCAGCCTTTGACATCA
BPIFB2-2:
ACCTGATGCCATGCATCGCACACCTCCACCCTTCGCAACTACCTCTCTTCCTGTAACTATTCTAGCCAGCCTTTGACATCA
BPIFB2-3:
ACCTGATGCCATGCATCGCATCTCATAACAAATCTTCATAATCTTCTATAACACTGTCAATCTAGCCAGCCTTTGACATCA
BPIFB2-4:
ACCTGATGCCATGCATCGCACATCTATATAGAATATATTCAATCCTCTCTCTTATATGCATCTAGCCAGCCTTTGACATCA
BPIFB2-5:
ACCTGATGCCATGCATCGCACTATCAACGACTCTCACGCAACGCTCTTCTTACTACATATACTAGCCAGCCTTTGACATCA
BPIFB2-6:
ACCTGATGCCATGCATCGCACCATTCATATCACGAACACCCATTTATACTATATACTACCACTAGCCAGCCTTTGACATCA
BPIFB2-7:
ACCTGATGCCATGCATCGCACGCACTAATATCCTTCATCCCTACTAATAACTCTTTTAACCCTAGCCAGCCTTTGACATCA
BPIFB2-8:
ACCTGATGCCATGCATCGCACACACTCATATTTATACACCAATATCATCTCACTCTATCTCCTAGCCAGCCTTTGACATCA
BPIFB2-9:
ACCTGATGCCATGCATCGCAAACCAAGAATGTCTTACGACATCTCGCCTACTATCCTATATCTAGCCAGCCTTTGACATCA
BPIFB2-10:
ACCTGATGCCATGCATCGCACACATAACAATCTATTATTCCTCTATCCGCTCTTATATTCCCTAGCCAGCCTTTGACATCA
BPIFB2-11:
ACCTGATGCCATGCATCGCAAACCAGTCTACTCAACCGACCACCTCAATATCATTCTCATCCTAGCCAGCCTTTGACATCA
BPIFB2-12:
ACCTGATGCCATGCATCGCACTACCTATACAATACTTACTGTCTGCTTCTACTTCTATTATCTAGCCAGCCTTTGACATCA
BPIFB2-13:
ACCTGATGCCATGCATCGCATATACTCGCAATCACTATATAAGACCACATAAATCACTTTACTAGCCAGCCTTTGACATCA
BPIFB2-14:
ACCTGATGCCATGCATCGCACTAACCCAATATTATTAACCGCACCACTCACATAAATCAAGCTAGCCAGCCTTTGACATCA
BPIFB2-15:
ACCTGATGCCATGCATCGCATATCAACTCTCATATATTCTTCACATCCCTACCCTTGTTACCTAGCCAGCCTTTGACATCA
BPIFB2-16:
ACCTGATGCCATGCATCGCAACAACATCTATCTACTTATCAACTTCAAAACTTTCATATTACTAGCCAGCCTTTGACATCA
实施例3蛋白结合适配子的性能测定
将适配子分别取2.0μg,用牛小肠碱性磷酸酶(CIP)37℃消化lh,纯化回收去磷酸化的RNA;通过T4多核苷酸激酶标记[γ-32P]ATP于去磷酸化的RNA分子末端。10nmol放射性标记的适配子分别与不同浓度(1-200nM)的BPIFB237℃孵育30min,各组反应液经硝酸纤维素膜滤过,洗涤滤膜,干燥滤膜,液闪计数仪测定滤膜上残留的放射量,同一样品平行做两次测定。计算各个适配子与目的蛋白的解离常数。结果如下:
实施例4所述适配子特异性分析以及稳定性分析
分别采用人血白蛋白,免疫血清球蛋白,霍乱弧菌VgrG3C蛋白,大肠杆菌外膜蛋白A,COCH蛋白,BPIFB2蛋白,与16条适配子进行特异性检测,经过结合试验发现,这些适配子都不与这些蛋白相结合,而只与BPIFB2蛋白结合保持较高的特异性。
将所述的适配子,取0.2ug,分别置于常温的血清、水溶液中,放置三周。通过RT-PCR检测,发现三周的放置其结构稳定,没有被降解。
实施例5所述适配子疾病的诊断
取11个口腔癌患者和4个正常人的唾液分泌物,使用生理盐水稀释,获得目标样本。
将16个偶联有标记的适配子分别与11个患者以及4个正常人的分泌物混合30min,通过生物素分离,定量分析其中的BPIFB2蛋白的含量,通过分析发现,11个口腔癌患者中BPIFB2蛋白的含量显著增加,超过了规定的阈值。达到了口腔癌的诊断标准。由此可见,其诊断效果较好。
以上仅为本发明的优选实施例而已,并不用于限制本发明,对于本领域的技术人员来说,凡在本发明的精神和原则之内所做的任何修改、等同替换、改进等,均应包含在本发明的保护范围之内。
序列表
〈110〉陈博
〈120〉一种口腔癌特异性检测的试剂盒
〈160〉15
〈210〉1
〈211〉 81
〈212〉DNA
〈213〉人工序列
〈400〉BPIFB2-1
ACCTGATGCCATGCATCGCATCACACGATCATCACACCGCTCCGCCTTCCAACTACCGCTC CTAGCCAGCCTTTGACATCA
〈210〉2
〈211〉 81
〈212〉DNA
〈213〉人工序列
〈400〉BPIFB2-2
ACCTGATGCCATGCATCGCACACCTCCACCCTTCGCAACTACCTCTCTTCCTGTAACTATT CTAGCCAGCCTTTGACATCA
〈210〉3
〈211〉 81
〈212〉DNA
〈213〉人工序列
〈400〉BPIFB2-3
ACCTGATGCCATGCATCGCATCTCATAACAAATCTTCATAATCTTCTATAACACTGTCAAT CTAGCCAGCCTTTGACATCA
〈210〉4
〈211〉 81
〈212〉DNA
〈213〉人工序列
〈400〉BPIFB2-4
ACCTGATGCCATGCATCGCACATCTATATAGAATATATTCAATCCTCTCTCTTATATGCAT CTAGCCAGCCTTTGACATCA
〈210〉5
〈211〉 81
〈212〉DNA
〈213〉人工序列
〈400〉BPIFB2-5
ACCTGATGCCATGCATCGCACTATCAACGACTCTCACGCAACGCTCTTCTTACTACATATA CTAGCCAGCCTTTGACATCA
〈210〉6
〈211〉 81
〈212〉DNA
〈213〉人工序列
〈400〉BPIFB2-6
ACCTGATGCCATGCATCGCACCATTCATATCACGAACACCCATTTATACTATATACTACCA CTAGCCAGCCTTTGACATCA
〈210〉7
〈211〉 81
〈212〉DNA
〈213〉人工序列
〈400〉BPIFB2-7
ACCTGATGCCATGCATCGCACGCACTAATATCCTTCATCCCTACTAATAACTCTTTTAACC CTAGCCAGCCTTTGACATCA
〈210〉8
〈211〉 81
〈212〉DNA
〈213〉人工序列
〈400〉BPIFB2-8
ACCTGATGCCATGCATCGCACACACTCATATTTATACACCAATATCATCTCACTCTATCTC CTAGCCAGCCTTTGACATCA
〈210〉9
〈211〉 81
〈212〉DNA
〈213〉人工序列
〈400〉BPIFB2-9
ACCTGATGCCATGCATCGCAAACCAAGAATGTCTTACGACATCTCGCCTACTATCCTATAT CTAGCCAGCCTTTGACATCA
〈210〉10
〈211〉 81
〈212〉DNA
〈213〉人工序列
〈400〉BPIFB2-10
ACCTGATGCCATGCATCGCACACATAACAATCTATTATTCCTCTATCCGCTCTTATATTCC CTAGCCAGCCTTTGACATCA
〈210〉11
〈211〉 81
〈212〉DNA
〈213〉人工序列
〈400〉BPIFB2-11
ACCTGATGCCATGCATCGCAAACCAGTCTACTCAACCGACCACCTCAATATCATTCTCATC CTAGCCAGCCTTTGACATCA
〈210〉12
〈211〉 81
〈212〉DNA
〈213〉人工序列
〈400〉BPIFB2-12
ACCTGATGCCATGCATCGCACTACCTATACAATACTTACTGTCTGCTTCTACTTCTATTAT CTAGCCAGCCTTTGACATCA
〈210〉13
〈211〉 81
〈212〉DNA
〈213〉人工序列
〈400〉BPIFB2-13
ACCTGATGCCATGCATCGCATATACTCGCAATCACTATATAAGACCACATAAATCACTTTA CTAGCCAGCCTTTGACATCA
〈210〉14
〈211〉 81
〈212〉DNA
〈213〉人工序列
〈400〉BPIFB2-14
ACCTGATGCCATGCATCGCACTAACCCAATATTATTAACCGCACCACTCACATAAATCAAG CTAGCCAGCCTTTGACATCA
〈210〉15
〈211〉 81
〈212〉DNA
〈213〉人工序列
〈400〉BPIFB2-15
ACCTGATGCCATGCATCGCATATCAACTCTCATATATTCTTCACATCCCTACCCTTGTTAC CTAGCCAGCCTTTGACATCA
〈210〉16
〈211〉 81
〈212〉DNA
〈213〉人工序列
〈400〉BPIFB2-16
ACCTGATGCCATGCATCGCAACAACATCTATCTACTTATCAACTTCAAAACTTTCATATTA CTAGCCAGCCTTTGACATCA

Claims (3)

1.一种用于口腔癌检测的试剂盒,其含有能特异性与BPIFB2蛋白结合的核酸适体。
2.如权利要求1所述的试剂盒,其特征在于:所述核酸适体序列为SEQ ID No:3所示。
3. 一种检测口腔癌的方法,其特征在于利用权利要求1-2任一项所述的试剂盒。
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