CN115747221B - Hcg-52及其在特异识别人绒毛促性腺激素中的应用 - Google Patents
Hcg-52及其在特异识别人绒毛促性腺激素中的应用 Download PDFInfo
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Abstract
本发明属于生物医药领域,具体涉及HCG‑52及其在特异识别人绒毛促性腺激素中的应用。具体地,所述核酸适配体的序列如SEQ ID NO.1或SEQ ID NO.2所示。
Description
技术领域
本发明属于生物医药领域,具体涉及HCG-52及其在特异识别人绒毛促性腺激素中的应用。
背景技术
人绒毛膜促性腺激素(human chorionic gonadotropin,hcg)是一种由胎盘绒毛膜囊泡的滋养层细胞分泌的糖蛋白激素,由α亚基和β亚基两条不同的亚基和244种氨基酸组成,分子量约为36.7kda。在结构上,α亚基与很多激素如促甲状腺激素、卵泡刺激素等相似,而β亚基是hcg特有的,故临床上hcg检测主要是利用β亚基的特异性。胎盘可产生hcg,滋养层细胞瘤、含滋养层组织的生殖细胞瘤以及一些非滋养层细胞瘤也可产生hcg。妊娠妇女血清中主要含有完整分子的hcg,其浓度会在怀孕早期呈指数型增长,并与时间有关,这对维持妊娠起着重要作用。
若hcg数值的变化不规律,特定时间过高或者过低,说明妊娠异常。检测结果的异常偏高提示绒毛膜癌、葡萄胎或多胎妊娠可能,检测结果偏低则提示先兆/早期流产、异位妊娠、妊娠中毒或胎儿宫内死亡。结合甲胎蛋白检测和准确的孕龄、孕妇体重等其他参数,检测hcg+β有助于在孕中期评价21三体综合征的风险,21三体孕妇的血清甲胎蛋白浓度降低,而母体血清hcg+β浓度可达到正常中位值的两倍。监测人绒毛膜促性腺激素水平可以预测妊高征的发生,对妊娠期高血压疾病的病程诊断有着重要的指导意义。hcg也是一种重要的血清和尿液肿瘤标志物,与妊娠无关的hcg浓度升高还可见于生殖细胞、卵巢、膀胱、胰腺、胃、肺和肝脏肿瘤病人。目前常用的检测方法有:胶乳集抑制试验和血凝抑制试验、放射免疫试验(RIA)、吸附试验(ELISA)、单克隆抗体胶体金试验。
指数富集的配基系统进化技术,简称SELEX(Systematic volution of LigandsbyEXponential Enrichment)技术,是近十几年兴起并迅速得到发展的高通量生物文库筛选技术。应用大容量的随机寡核苷酸文库(ssDNA文库和RNA文库),结合PCR体外扩增技术,以指数级富集与靶分子特异结合的寡核苷酸,经过反复的体外筛选、扩增,最终获得的核酸适配体(aptamer)基于空间结构与靶分子呈高特异性和高亲和力的结合。
核酸适配体具有精确识别、无免疫原性、易体外合成与修饰等优点,又称为“人工替代抗体”,在基础医学、临床诊断与新药研发等方面具有广阔的应用前景。
发明内容
本发明通过SELEX技术以人绒毛膜促性腺激素作为靶标,筛选获得了一组特异识别人绒毛膜促性腺激素(human chorionic gonadotropin,hcg)的核酸适配体HCG-5 2及其在识别人绒毛膜促性腺激素中的应用。所述核酸适配体是DNA序列,不仅可以直接用于诊断,还可以作为分子探针构建生物检测传感器等。经鉴定该核酸适配体及其截短序列均能够特异识别人绒毛膜促性腺激素,而不结合其它无关蛋白(BSA蛋白),对照核酸序列与人绒毛膜促性腺激素均无结合。
具体技术方案如下:
一方面,本发明提供了一种核酸适配体,所述核酸适配体的序列如SEQ IDNO.1或2所示。
优选地,其中SEQ ID NO.2(又称为HCG-52S,或“截短体”)所示核酸适配体是SEQID NO.1所示核酸适配体的核心区域,SEQ ID NO.2所示核酸适配体具有特异性结合hcg的功能,在SEQ ID NO.2所示核酸适配体的任意一端(3’端、5’端)添加任意数量的核苷酸后,依然会保持其与hcg特异性结合的功能。
或者,对于核酸适配体而言,通过使用MFOLD程序来预测二级结构,或者通过使用X射线分析或NMR分析来预测立体结构,从而可以在一定程度上预测哪些核苷酸可以被替换或缺失,以及在哪里可以插入新的核苷酸。可以容易地化学合成具有新序列的预测的适配体,并且可以使用现有的测定系统来确认该适配体是否保持活性。
优选地,所述核酸适配体上可以带有额外的一个或多个化学基团修饰,具体地,所述化学修饰可以发生在碱基或核糖上。在本说明书中,“碱基”是指构成核酸的腺嘌呤(A)、鸟嘌呤(G)、胞嘧啶(C)、尿嘧啶(U)或胸腺嘧啶(T)的任一者。
本发明所述的化学基团包括,但不限于H、D、氧代(=O)、F、Cl、Br、-OH、-CN、-NO2、-NRcRd、-C(=O)R9、-OC(=O)R9、-C(=O)OR9a、-S(=O)0-2R9、-OS(=O)1-2R9、-S(=O)1-2OR9a、-N(R10a)C(=O)R10、-C(=O)NR10aR10、-OC(=O)NR10aR10、-N(R10a)S(=O)1-2R10、-S(=O)1- 2NR10aR10、-N(R10a)C(=O)NR10aR10、C1-6烷基、C2-6烯基、C2-6炔基、C1-6卤代烷基、C1-6羟基烷基、C1-6氨基烷基、氰基取代的C1-6烷基、C1-6烷氧基、C1-6烷基氨基、C3-8环烷基、C3-8环烷基C1-6烷基、C2-7杂环基、C2-7杂环基C1-6烷基、C6-12芳基、C6-12芳基C1-6烷基、C1-9杂芳基、或C1-9杂芳基C1-6烷基;其中所述各C3-8环烷基、C3-8环烷基C1-6烷基、C2-7杂环基、C2-7杂环基C1-6烷基、C6-12芳基、C6-12芳基C1-6烷基、C1-9杂芳基、C1-9杂芳基等。
如本发明所述,术语“核酸适配体”是指一种能和靶分子特异性结合的单链寡核苷酸,本发明提供的核酸适配体与hcg之间的结合反应是非随机的。本发明中,所述靶分子是hcg。
如本发明所述,术语“hcg”指的是uman chorionic gonadotropin,人绒毛膜促性腺激素,人绒毛膜促性腺激素几乎仅由滋养细胞产生,最主要由胎盘滋养细胞分泌,男性和未受孕女性的垂体也可少量分泌。该激素的生理作用包括促进排卵和胚泡植入、维持黄体寿命和保胎,不仅能促进性腺的发育和性激素的分泌,还能促进第二性征的发育、刺激甲状腺的活性及保护滋养层不受免疫攻击。该指标可帮助了解胎盘功能,对于正常妊娠以及妊娠期特有疾病、胎儿先天性缺陷或疾病的诊断或筛查有重要意义。hCG检查属于生殖系统激素检查,包括定性和定量检查两种。
另一方面,本方面提供了可检测标记物标记的核酸适配体。
优选地,所述标记可以是通过化学键连接或物理吸附。
优选地,所述可检测标记物包括放射性标记物、化学发光标记物、荧光标记物、亲和素、生物素、地高辛、或酶。具体地,合适的酶包括辣根过氧化物酶、碱性磷酸酶、β半乳糖苷酶或乙酰胆碱酯酶;合适的辅基包括抗生蛋白链菌素、抗生物素蛋白和生物素;合适的荧光物质包括但不限于FAM(羧基荧光素,Carboxy fluorescein,绿色荧光)、FITC(异硫氰酸荧光素,Fluoresceinisothiocyanate)、TET(四氯-6-羧基荧光素,Tetrachlorofluorescein)、HEX(六氯-6-甲基荧光素,Hexachloro fluorescein);罗丹明类型的标记,包括TAMRA;丹磺酰;丽丝胺;花菁;藻红蛋白;德克萨斯红;及其类似物。
如本发明具体实施例所使用的可检测标记物是生物素,其标记核酸适配体的方法是本领域常规的。
优选地,所述核酸适配体、可检测标记物标记的核酸适配体在日常储存中可固定于合适的固相载体上,以便于更方便、可视化的检测、鉴定hcg;或者所述核酸适配体、可检测标记物标记的核酸适配体也可以存储与合适的液体中以保持其稳定性,例如水、缓冲液。
另一方面,本发明提供给了一种检测hcg的方法,所述方法包括将待测物与本发明所述核酸适配体或可检测标记物标记的核酸适配体接触。
优选地,所述待测物包括采集自人体的样品。
优选地,所述待测物中疑似含有hcg。
优选地,所述样品包括外周血、组织、血液、血清、血浆、尿液、唾液、精液、乳汁、脑脊髓液、泪液、痰、粘液、淋巴、胞液、腹水、胸膜积液、羊水、膀胱冲洗液和支气管肺泡灌洗液。
优选地,所述样品包括尿液和血液。
优选地,所述方法是非诊断目的的。
优选地,所述接触保持至少5、10、15、20、30、40分钟或更长时间。
优选地,所述方法还包括使用报告集团使检测结果可视化的步骤。
优选地,所述核酸适配体是经过变性处理的。
更优选地,所述变性处理的具体步骤是:将核酸适配体溶解在缓冲液中,100℃变性后冷却;
优选地,所述缓冲液的成分是50mM HEPES,100mM NaCl,2mM MgCl2,5mMKCl,1mMCaCl2。
另一方面,本发明还提供了本发明核酸适配体、可检测标记物标记的核酸适配体在特异性结合hcg、检测hcg中的应用。
优选地,所述特异性结合是在体外发生的、非诊断目的的。
优选地,所述检测包括定量检测或定性检测,所述定性检测的结果包括待测物中“存在”或“不存在”hcg。
优选地,所述检测通过以下任意方法实现:酶免疫测定、放射免疫测定、荧光免疫测定、Western印迹法、免疫组织化学染色法、细胞分选法。
另一方面,本发明还提供了本发明核酸适配体、可检测标记物标记的核酸适配体在制备诊断hcg相关症状的产品中的应用。
优选地,所述hcg相关症状包括受孕、绒毛膜癌、葡萄胎、多胎妊娠、先兆/早期流产、异位妊娠、妊娠中毒、胎儿宫内死亡、21三体综合征、妊娠期高血压疾病、生殖细胞肿瘤、卵巢肿瘤、膀胱肿瘤、胰腺肿瘤、胃肿瘤、肺肿瘤和肝脏肿瘤。
另一方面,本发明提供了一种靶向hcg的探针的制备方法,所述方法包括获得本发明所述核酸适配体,并使用可检测标记物标记所述核酸适配体。
本发明的适配体可以按照本说明书中的公开和本领域中本身已知的方法化学合成、生物合成,所述生物合成例如聚合酶链式反应(Polymerase ChainReaction,简称PCR)方法或其他恒温、变温地核酸扩增方法。
本说明书中提及的所有出版物、专利和专利申请均通过引用并入本文,如同每个单独的出版物、专利或专利申请都被具体和单独地指出通过引用结合于此。其程度就如同每个单独出版物、专利或专利申请具体地和单独地表示为通过引用并入。
附图说明
图1是HCG-52特异结合人绒毛促性腺激素hCG的EMSA实验结果图。
图2是酶联法检测的结果统计图。
图3是梯度浓度的核酸适配体进行酶联法检测的结果统计图。
具体实施方式
下面结合实施例对本发明做进一步的说明,以下所述,仅是对本发明的较佳实施例而已,并非对本发明做其他形式的限制,任何熟悉本专业的技术人员可能利用上述揭示的技术内容加以变更为同等变化的等效实施例。凡是未脱离本发明方案内容,依据本发明的技术实质对以下实施例所做的任何简单修改或等同变化,均落在本发明的保护范围内。
实施例1、EMSA实验验证核酸适配体的特异性
1、实验材料
(1)人绒毛膜促性腺激素:华阳正龙货号:201215
(2)牛血清白蛋白(BSA):北京中生奥邦生物公司货号:01.10001D
(3)生物素标记的核酸适配体:生工合成,具体序列如表1所示
(4)HRP酶:Solarbio货号898800
表1、核酸适配体的序列
2、实验方法
2.1分别将一定浓度的Bio-HCG-52适配体和无关对照序列Bio-AAAA溶解于合适体积的缓冲液中(50mM HEPES,100mM NaCl,2mM MgCl2,5mM KCl,1mM CaCl2),于100℃变性5min后立即置于冰上充分冷却;
2.2将经过变性处理后的Bio-HCG-52与人绒毛促性腺激素hCG蛋白(或者无关蛋白BSA)在37℃共孵育40min;
2.3适配体与人绒毛促性腺激素hCG的共同孵育体系加入10×DNA上样缓冲液,6%天然PAGE胶电泳分离;
2.4卸胶后转模,紫外交联2min后封闭液封闭30min;
2.5将膜放入按照1:1000稀释好的HRP酶中,室温孵育40min,将膜取出并洗膜2次,每次10min;
2.6加TMB底物显色并留图。
3、实验结果
如图1所示,EMSA实验证明核酸适配体HCG-52特异结合人绒毛促性腺激素hCG,而不结合其它无关蛋白(BSA蛋白)。
实施例2、酶联法验证核酸适配体的特异性
1、实验材料
同实施例1。
2、实验方法
2.1将一定量的人绒毛促性腺激素hCG融于pH 9.7的碳酸盐缓冲液中,按照100μl/孔加入到酶联条中,4℃包被过夜;
2.2弃包被液,每孔加入100μl含2%BSA封闭液,室温封闭60min;
2.3分别将一定浓度的Bio-HCG-52/Bio-HCG-52S适配体和无关对照序列Bio-AAAA溶解于合适体积的缓冲液中(50mM HEPES,100mM NaCl,2mMMgCl2,5mM KCl,1mM CaCl2),于100℃变性5min后立即置于冰上充分冷却;
2.4将经过变性处理后的生物素标记的Bio-HCG-52/Bio-HCG-52S和无关对照序列Bio-AAAA加入酶联条中,使核酸适配体与包被的人绒毛促性腺激素hCG蛋白共同孵育37℃30min;
2.5弃去孔内液体,每孔用200μl的洗涤液洗涤,重复洗涤3次,最后一次洗涤后要把孔内液体完全甩干;
2.6每孔加入100μl按照1:100稀释好的HRP酶,室温孵育40min,弃去孔内液体,洗板5次,方法同上;
2.7每孔加入100μl TMB显色底物,37℃避光显色,当有明显颜色变化时,加10μl终止液,酶联仪读数。
3、实验结果
酶联仪读数结果统计如图2所示,ELISA实验证明核酸适配体HCG-52及其截短序列HCG-52S均可特异结合人绒毛促性腺激素hCG。
实施例3、浓度梯度结合实验
1、实验材料
同实施例1。
2、实验方法
2.1将一定量的人绒毛促性腺激素hCG融于pH 9.7的碳酸盐缓冲液中,按照100μl/孔加入到酶联条中,4℃包被过夜;
2.2弃包被液,每孔加入100μl含2%BSA封闭液,室温封闭60min;
2.3分别将0μg、0.5μg、1μg、2μg、3μg的Bio-HCG-52/Bio-HCG-52S适配体和无关对照序列Bio-AAAA溶解于合适体积的缓冲液中(50mMHEPES,100mM NaCl,2mM MgCl2,5mMKCl,1mM CaCl2),于100℃变性5min后立即置于冰上充分冷却;
2.4将经过变性处理后的生物素标记的Bio-HCG-52/Bio-HCG-52S和无关对照序列Bio-AAAA加入酶联条中,使核酸适配体与包被的人绒毛促性腺激素hCG蛋白共同孵育37℃30min;
2.5弃去孔内液体,每孔用200μl的洗涤液洗涤,重复洗涤3次,最后一次洗涤后要把孔内液体完全甩干;
2.6每孔加入100μl按照1:100稀释好的HRP酶,室温孵育40min,弃去孔内液体,洗板5次,方法同上;
2.7每孔加入100μl TMB显色底物,37℃避光显色,当有明显颜色变化时,加10μl终止液,酶联仪读数。
3、实验结果
酶联仪读数结果统计如图3所示,ELISA实验证明核酸适配体HCG-52及其截短序列HCG-52S与人绒毛促性腺激素的结合有浓度梯度,而对照序列AAAA则没有。
Claims (12)
1.一种核酸适配体,所述核酸适配体的序列如SEQ ID NO.1或SEQ ID NO.2所示。
2.可检测标记物标记的核酸适配体,所述核酸适配体的序列如SEQ ID NO.1或SEQ IDNO.2所示,所述可检测标记物包括放射性标记物、化学发光标记物、荧光标记物、亲和素、生物素、地高辛或酶。
3.一种检测hcg的方法,所述方法包括将待测物与权利要求1所述核酸适配体或权利要求2所述可检测标记物标记的核酸适配体接触,所述方法是非诊断目的的。
4.如权利要求3所述的方法,所述待测物包括采集自人体的样品。
5.如权利要求4所述的方法,所述样品包括组织、血液、血清、血浆、尿液、唾液、精液、乳汁、脑脊髓液、泪液、痰、淋巴、胞液、腹水、胸膜积液、羊水、膀胱冲洗液和支气管肺泡灌洗液。
6.如权利要求5所述的方法,所述样品包括尿液和血液。
7.如权利要求3所述的方法,所述核酸适配体是经过变性处理的,所述变性处理的具体步骤是:将核酸适配体溶解在缓冲液中,100℃变性后冷却。
8.权利要求1所述核酸适配体或权利要求2所述可检测标记物标记的核酸适配体在制备特异性结合hcg、检测hcg的产品中的应用。
9.权利要求1所述核酸适配体或权利要求2所述可检测标记物标记的核酸适配体在制备诊断hcg相关症状的产品中的应用。
10.如权利要求9所述应用,所述hcg相关症状包括受孕、绒毛膜癌、葡萄胎、多胎妊娠、先兆/早期流产、异位妊娠、妊娠中毒、胎儿宫内死亡、21三体综合征、妊娠期高血压疾病、生殖细胞肿瘤、卵巢肿瘤、膀胱肿瘤、胰腺肿瘤、胃肿瘤、肺肿瘤和肝脏肿瘤。
11.一种靶向hcg的探针的制备方法,所述方法包括合成权利要求1所述核酸适配体,并使用可检测标记物标记所述核酸适配体。
12.如权利要求11所述制备方法,所述合成包括化学合成或生物合成。
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