CN115925866A - 一种用于乳腺癌检测的单克隆抗体及其试剂盒 - Google Patents
一种用于乳腺癌检测的单克隆抗体及其试剂盒 Download PDFInfo
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Abstract
本发明一方面公开了一种乳腺癌标志物PMEPA1的抗原表位多肽。本发明还公开了一种能特异性结合所述的PMEPA1抗原表位多肽的单克隆抗体。本发明还公开了一种用于乳腺癌检测的试剂盒。本发明基于筛选抗原性好、抗原表位多的序列设计两条彼此分开的抗原表位多肽，再此基础上筛选合适的单克隆抗体，以用于对PMEPA1蛋白的检测；该方法具有以下优点：1)可以提高筛选单克隆抗体的成功率；2)可以省略单克隆抗体的配对检验；3)可以提高单克隆抗体的特异性。本发明的试剂盒具有良好的特异性和敏感性，适用于PMEPA1蛋白的检测。
Description
技术领域
本发明属于生物技术领域，具体涉及一种用于乳腺癌检测的单克隆抗体及其试剂盒。
背景技术
乳腺癌是乳腺上皮细胞在多种致癌因子的作用下，发生增殖失控的现象。疾病早期常表现为***肿块、***溢液、腋窝***肿大等症状，晚期可因癌细胞发生远处转移，出现多器官病变，直接威胁患者的生命。
近年研究表明，乳腺癌的发生发展与蛋白的异常表达和信号通路的异常激活密切相关。有研究发现，在乳腺癌细胞中雄激素诱导的***跨膜蛋白1(prostatetransmembrane protein,androgen induced 1，PMEPA1)基因较正常细胞出现5～10倍的过表达。目前已证实PMEPA1能通过参与调控多条信号通路,对恶性肿瘤细胞的增殖、侵袭、转移等多种生物学行为产生重要影响,并且对恶性肿瘤患者的预后起到一定的预测作用。
发明内容
为了弥补现有技术的不足，本发明的目的之一，提供一种乳腺癌标志物PMEPA1的抗原表位序列；本发明的目的之二，提供了一种针对PMEPA1抗原表位序列的单克隆抗体。
因此，本发明一方面公开了一种乳腺癌标志物PMEPA1的抗原表位多肽，所述的PMEPA1抗原表位多肽的氨基酸序列为：抗原表位多肽1：LMGVNSTAAAAAGQPNVSCTCNCKRSLFQSM；抗原表位多肽2：LGGPCPPSSNSGISATCYGSGGRMEGPPPTYS。
另一方面，本发明还公开了一种能特异性结合所述的PMEPA1抗原表位多肽的单克隆抗体，所述的编码特异性结合抗原表位多肽1的单克隆抗体1的重链可变区的核苷酸序列如SEQ ID NO.1所示，所述的编码特异性结合抗原表位多肽1的单克隆抗体1的轻链可变区的核苷酸序列如SEQ ID NO.2所示；所述的编码特异性结合抗原表位多肽2的单克隆抗体2的重链可变区的核苷酸序列如SEQ ID NO.3所示，所述的编码特异性结合抗原表位多肽2的单克隆抗体2的轻链可变区的核苷酸序列如SEQ ID NO.4所示。
优选地，本发明所述的单克隆抗体1的重链可变区序列包含CDR-H1、CDR-H2和CDR-H3，所述的重链可变区的CDR-H1、CDR-H2和CDR-H3的序列分别为：
CDR-H1：NSMN；
CDR-H2：EIRLKSNNYVTHYAESVKG；
CDR-H3：IYSPFAY；
所述的单克隆抗体1的轻链可变区序列包含CDR-L1、CDR-L2和CDR-L3，所述的轻链可变区的CDR-L1、CDR-L2和CDR-L3的序列分别为：
CDR-L1：PPSSEKASIDVSTIVS；
CDR-L2：SATYRYI；
CDR-L3：QQHYSSPST。
优选地，本发明所述的单克隆抗体1的重链可变区序列还包含FR-H1、FR-H2、FR-H3和FR-H4，所述的重链可变区的FR-H1、FR-H2、FR-H3和FR-H4的序列分别为：
FR-H1：ESGGGLVGSQPGGSMKLSCVASGFTIS；
FR-H2：WVRQSPEKGLDWVA；
FR-H3：RFTISRDDSKNSVYLQSMNNLRPEGSGIYYCTP；
FR-H4：WGQGTLVTVSA；
所述的单克隆抗体1的轻链可变区序列还包含FR-L1、FR-L2、FR-L3和FR-L4，所述的轻链可变区的FR-L1、FR-L2、FR-L3和FR-L4的序列分别为：
FR-L1：DIVMSQSHKFDSTSVGDRVSITC；
FR-L2：WYQQKPGQSPKLLHY；
FR-L3：GVPDRFTASGSGTWFTFTISSVQAEDLAVYYC；
FR-L4：FGAGTCPTKLEL。
优选地，本发明所述的单克隆抗体2的重链可变区序列包含CDR-H1、CDR-H2和CDR-H3，所述的重链可变区的CDR-H1、CDR-H2和CDR-H3的序列分别为：
CDR-H1：DYYMY；
CDR-H2：ISNGGGTTYSPTVKG；
CDR-H3：HEHGYTPYVAY；
所述的单克隆抗体2的轻链可变区序列包含CDR-L1、CDR-L2和CDR-L3，所述的轻链可变区的CDR-L1、CDR-L2和CDR-L3的序列分别为：
CDR-L1：KSSQSLYSSNQKNYSWTLA；
CDR-L2：WASTRES；
CDR-L3：QQCYSYPRT。
优选地，本发明所述的单克隆抗体2的重链可变区序列还包含FR-H1、FR-H2、FR-H3和FR-H4，所述的重链可变区的FR-H1、FR-H2、FR-H3和FR-H4的序列分别为：
FR-H1：CEKLVESGGGLVQPGGTPLKLSCATSGFTFS；
FR-H2：WTPVRQTPEKRLEWVA；
FR-H3：RFTSRDNAKTLYLQSRKSEDTAMTPYYCAR；
FR-H4：WGQGTLVTVSA；
所述的单克隆抗体2的轻链可变区序列还包含FR-L1、FR-L2、FR-L3和FR-L4，所述的轻链可变区的FR-L1、FR-L2、FR-L3和FR-L4的序列分别为：
FR-L1：DIVMSQSPSSTSLAVSVGEKVTMSC；
FR-L2：WYQQKPGQSPKLLIY；
FR-L3：GVPDRFTGSGSGTDFTTISSVKAEDLSWTAVYYC；
FR-L4：FGGGTKLEIK。
再一方面，本发明还公开了一种用于乳腺癌检测的试剂盒，所述试剂盒包括有效量的权利要求2所述的单克隆抗体1和单克隆抗体2；以及配套的检测试剂。
再一方面，本发明还公开了一种所述的PMEPA1抗原表位多肽在制备乳腺癌诊断试剂中的应用。
本发明基于筛选抗原性好、抗原表位多的序列(如实施例1所述)设计两条彼此分开的抗原表位多肽，再次基础上筛选合适的单克隆抗体，以用于对PMEPA1蛋白的检测。该方法具有以下优点：1)可以提高筛选单克隆抗体的成功率(避免使用全蛋白筛选时的无效抗体)；2)可以省略单克隆抗体的配对检验；3)可以提高单克隆抗体的特异性(抗原表位为高度特异性的抗原位点，能被多个单克隆抗体特异性结合)。
在筛选的单克隆抗体的基础上，形成了检测PMEPA1蛋白的试剂盒，该试剂盒具有良好的特异性和敏感性，适用于PMEPA1蛋白的检测。虽然本发明采用的检测方法为常规的ELISA方法，但是，本发明筛选的单克隆抗体同样可以应用于其他免疫学方法的检测，如胶体金检测、化学发光检测等等。
附图说明
图1PMEPA1蛋白跨膜区域预测分析结果。其中Inside表示胞内区，inside数值越大，表示该氨基酸位于胞内区的可能性越大；Outside表示胞外区，outside数值越大，表示该氨基酸位于胞外区的可能性越大；Transmembrane表示跨膜区，Transmembrane数值越大，表示该氨基酸在跨膜区的可能性越大。
图2PMEPA1蛋白二级结构预测分析结果。
具体实施方式
以下结合具体实施例来进一步说明本发明，但实施例并不对本发明做任何形式的限定。除非特别说明，本发明采用的试剂、方法和设备为本技术领域常规试剂、方法和设备。
除非特别说明，以下实施例所用试剂和材料均为市购。
实施例1：PMEPA1抗原表位多肽的设计与合成
从NCBI搜索PMEPA1，从中找到近年公开的PMEPA1序列，选择其中之一(NM_020182.5)作为本研究的目的蛋白，对该蛋白的跨膜区域(图1)和二级结构(图2)进行预测分析，选择其中两段作为PMEPA1的抗原表位多肽，并交公司合成多肽，以用于后续试验。其中抗原表位的设计遵循以下原则(参见中国发明专利2015106122558)：选择细胞膜蛋白表面区域或内部区域，避开跨膜区域；选择不形成a-helix的序列；两端的肽段比中间的排列合理；避免蛋白内部重复；避免同源性强的肽段。
经过设计，两个抗原表位多肽序列为：
抗原表位多肽1：LMGVNSTAAAAAGQPNVSCTCNCKRSLFQSM；
抗原表位多肽2：LGGPCPPSSNSGISATCYGSGGRMEGPPPTYS。
实施例2：抗PMEPA1抗原表位多肽单克隆抗体的制备
2.1动物免疫将实施例1制备的抗原表位多肽分别作为抗原，与等质量福氏完全佐剂充分乳化后背腹部皮下多点注射8周龄左右BALB/c雌性小鼠，0.2ml/只；间隔2周，取与一免等量抗原和等质量的福氏不完全佐剂充分乳化后，第二次腹腔注射0.2ml/只，过2周后用加倍剂量的抗原进行腹腔注射免疫，3天后取脾细胞进行细胞融合。
2.2细胞融合及杂交瘤细胞筛选采集小鼠脾脏研磨并分离脾细胞，使用PEG法将其与骨髓瘤Sp2/0细胞株融合，将融合后的细胞用HAT培养基重悬，均匀铺于96孔板内，每孔100μl，37℃，5％CO2培养。细胞培养箱中培养5天后，用HAT培养基换液一次，第10天用HT培养基换液，等到融合细胞覆盖孔底10～30％时。取培养上清，用PMEPA1抗原表位多肽包被的常规间接ELISA方法进行阳性克隆的检测。共获98个阳性孔，分别选择3个呈强阳性反应的细胞孔，进行3次有限稀释法细胞克隆，分别获得1株杂交瘤细胞，分别命名为1号(抗原表位多肽1免疫后筛选获得)和2号(抗原表位多肽2免疫后筛选获得)。经传代和多次冻存复苏后，细胞株均能良好生长，并稳定分泌抗体。经扩大培养后，用于腹水制备和液氮保存。
2.3单克隆抗体的腹水制备和纯化取8周龄左右BALB/c雌性小鼠经腹腔注射降植烷，0.3～0.5ml/只，7日后每只小鼠注射杂交瘤细胞1×106个。注射后7～10天可见小鼠腹部明显膨大，注射针头采取腹水，4℃8000rpm离心3min，收集上清液，即为单抗腹水。取1倍体积腹水，加入2倍体积醋酸盐缓冲液(0.06mol/L，pH值4.8)，混合均匀后在室温下边搅拌边加入辛酸(30μl/ml腹水)，4℃澄清2h小时，以4℃12000rpm离心20min，收集上清。再用50％饱和硫酸铵沉淀免疫球蛋白，4℃静置2小时，4℃3000rpm离心30min，收集沉淀。用2倍体积PBS溶解沉淀后用PBS透析过夜，即获得纯化的腹水抗体，置于-70℃保存备用。
2.4单克隆抗体分析提取杂交瘤细胞的总RNA并进行逆转录，将获得的cDNA保存于-15℃以下待用。设计特异性的巢式PCR引物，采用常规PCR方法扩增目的基因。其中，引物序列的设计按照文献(CN 111393525 B)进行。经过测序，单克隆抗体1(抗原表位多肽1免疫后制备)和单克隆抗体2(抗原表位多肽2免疫后制备)的可变区氨基酸序列信息见表1所示。
表1单克隆抗体氨基酸序列信息
实施例3：检测PMEPA1免疫学方法的建立及其试剂盒的制备
3.1试剂盒的制备为常规ELISA试剂盒制备方法，简述如下：
3.1.1酶标板制备使用单克隆抗体1包被酶标板(包被液为0.05M的碳酸盐缓冲液，pH9.6)，100ng/孔，0.1ml/孔，4℃包被过夜，洗涤3次后，使用含1％BSA的PBST封闭，37℃孵育2h，洗涤后存于-15℃以下保存备用；
3.1.2试剂盒检测方法使用样品稀释液(含1％ BSA的PBST缓冲液)将待检样品稀释50倍，混匀后加到包被的酶标板中，0.1ml/孔，37℃孵育1h；洗涤后，加入用样品稀释液1:10000倍稀释的HRP标记的单克隆抗体2(HRP标记为常规的改良高碘酸钠方法)，0.1ml/孔，37℃孵育30min；洗涤后，加入0.1ml TMB单组分显色液，37℃孵育10min；加入终止液(2M硫酸)，测定OD450nm值，当P/N值＞2.1时判为阳性，否则为阴性，其中P为样品OD450nm值，N为空白对照OD450nm值。
3.2试剂盒检测本研究从广东省某医院获取80名乳腺癌患者和80名非患者中的血液样本；使用3.1建立的试剂盒对其PMEPA1蛋白进行检测，结果显示，该试剂盒检测的80名乳腺癌患者均为阳性，80名非患者均为阴性，说明试剂盒具有良好的特异性和敏感性。
上述实施例为本发明较佳的实施方式，但本发明的实施方式并不受上述实施例的限制，其他的任何未背离本发明的精神实质与原理下所作的改变、修饰、替代、组合、简化，均应为等效的置换方式，都包含在本发明的保护范围之内。
Claims (8)
1.一种乳腺癌标志物PMEPA1的抗原表位多肽，其特征在于，所述的PMEPA1抗原表位多肽的氨基酸序列为：
抗原表位多肽1：LMGVNSTAAAAAGQPNVSCTCNCKRSLFQSM；
抗原表位多肽2：LGGPCPPSSNSGISATCYGSGGRMEGPPPTYS。
2.一种能特异性结合权利要求1所述的PMEPA1抗原表位多肽的单克隆抗体，其特征在于，所述的编码特异性结合抗原表位多肽1的单克隆抗体1的重链可变区的核苷酸序列如SEQ ID NO.1所示，所述的编码特异性结合抗原表位多肽1的单克隆抗体1的轻链可变区的核苷酸序列如SEQ ID NO.2所示；所述的编码特异性结合抗原表位多肽2的单克隆抗体2的重链可变区的核苷酸序列如SEQ ID NO.3所示，所述的编码特异性结合抗原表位多肽2的单克隆抗体2的轻链可变区的核苷酸序列如SEQ ID NO.4所示。
3.根据权利要求2所述的单克隆抗体，其特征在于，所述的单克隆抗体1的重链可变区序列包含CDR-H1、CDR-H2和CDR-H3，所述的重链可变区的CDR-H1、CDR-H2和CDR-H3的序列分别为：
CDR-H1：NSMN；
CDR-H2：EIRLKSNNYVTHYAESVKG；
CDR-H3：IYSPFAY；
所述的单克隆抗体1的轻链可变区序列包含CDR-L1、CDR-L2和CDR-L3，所述的轻链可变区的CDR-L1、CDR-L2和CDR-L3的序列分别为：
CDR-L1：PPSSEKASIDVSTIVS；
CDR-L2：SATYRYI；
CDR-L3：QQHYSSPST。
4.根据权利要求2所述的单克隆抗体，其特征在于，所述的单克隆抗体1的重链可变区序列还包含FR-H1、FR-H2、FR-H3和FR-H4，所述的重链可变区的FR-H1、FR-H2、FR-H3和FR-H4的序列分别为：
FR-H1：ESGGGLVGSQPGGSMKLSCVASGFTIS；
FR-H2：WVRQSPEKGLDWVA；
FR-H3：RFTISRDDSKNSVYLQSMNNLRPEGSGIYYCTP；
FR-H4：WGQGTLVTVSA；
所述的单克隆抗体1的轻链可变区序列还包含FR-L1、FR-L2、FR-L3和FR-L4，所述的轻链可变区的FR-L1、FR-L2、FR-L3和FR-L4的序列分别为：
FR-L1：DIVMSQSHKFDSTSVGDRVSITC；
FR-L2：WYQQKPGQSPKLLHY；
FR-L3：GVPDRFTASGSGTWFTFTISSVQAEDLAVYYC；
FR-L4：FGAGTCPTKLEL。
5.根据权利要求2所述的单克隆抗体，其特征在于，所述的单克隆抗体2的重链可变区序列包含CDR-H1、CDR-H2和CDR-H3，所述的重链可变区的CDR-H1、CDR-H2和CDR-H3的序列分别为：
CDR-H1：DYYMY；
CDR-H2：ISNGGGTTYSPTVKG；
CDR-H3：HEHGYTPYVAY；
所述的单克隆抗体2的轻链可变区序列包含CDR-L1、CDR-L2和CDR-L3，所述的轻链可变区的CDR-L1、CDR-L2和CDR-L3的序列分别为：
CDR-L1：KSSQSLYSSNQKNYSWTLA；
CDR-L2：WASTRES；
CDR-L3：QQCYSYPRT。
6.根据权利要求2所述的单克隆抗体，其特征在于，所述的单克隆抗体2的重链可变区序列还包含FR-H1、FR-H2、FR-H3和FR-H4，所述的重链可变区的FR-H1、FR-H2、FR-H3和FR-H4的序列分别为：
FR-H1：CEKLVESGGGLVQPGGTPLKLSCATSGFTFS；
FR-H2：WTPVRQTPEKRLEWVA；
FR-H3：RFTSRDNAKTLYLQSRKSEDTAMTPYYCAR；
FR-H4：WGQGTLVTVSA；
所述的单克隆抗体2的轻链可变区序列还包含FR-L1、FR-L2、FR-L3和FR-L4，所述的轻链可变区的FR-L1、FR-L2、FR-L3和FR-L4的序列分别为：
FR-L1：DIVMSQSPSSTSLAVSVGEKVTMSC；
FR-L2：WYQQKPGQSPKLLIY；
FR-L3：GVPDRFTGSGSGTDFTTISSVKAEDLSWTAVYYC；
FR-L4：FGGGTKLEIK。
7.一种用于乳腺癌检测的试剂盒，其特征在于，所述试剂盒包括有效量的权利要求2所述的单克隆抗体1和单克隆抗体2；以及配套的检测试剂。
8.一种如权利要求2所述的单克隆抗体在制备乳腺癌诊断试剂中的应用。
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