CN107573414B - 人膀胱癌标志物ag-cd71及其抗体abc71和应用 - Google Patents

人膀胱癌标志物ag-cd71及其抗体abc71和应用 Download PDF

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CN107573414B
CN107573414B CN201710692872.2A CN201710692872A CN107573414B CN 107573414 B CN107573414 B CN 107573414B CN 201710692872 A CN201710692872 A CN 201710692872A CN 107573414 B CN107573414 B CN 107573414B
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bladder cancer
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李翀
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Abstract

本发明主要属于肿瘤免疫学技术领域，具体涉及一种新型膀胱癌标志物AG‑CD71，以及抗AG‑CD71的单克隆抗体ABC71。本发明所述人膀胱癌标志物AG‑CD71，其为异常糖基化的转铁蛋白受体TFRC，所述异常糖基化的转铁蛋白受体TFRC是指TFRC上带有作为抗原表位的糖结构Fuca1‑4(GlcNAcb1‑3)[6OSO3]GlcNAc；并提供一种针对所述的人膀胱癌标志物AG‑CD71的抗体，所述抗体为特异性抗人膀胱癌AG‑CD71的单克隆抗体ABC71，该单克隆抗体由保藏号为CGMCC No.14312的杂交瘤细胞株分泌。细胞和组织学水平证明：ABC71特异性识别人膀胱癌细胞及人膀胱癌组织。抗人膀胱癌的单克隆抗体ABC71，该抗体与人膀胱癌组织呈强阳性反应，而与人正常膀胱组织呈阴性反应。

Description

人膀胱癌标志物AG-CD71及其抗体ABC71和应用
技术领域
本发明主要属于肿瘤免疫学技术领域，具体涉及一种新型膀胱癌标志物AG-CD71，以及抗AG-CD71的单克隆抗体ABC71。细胞和组织学水平证明：ABC71特异性识别人膀胱癌细胞及人膀胱癌组织。本发明还涉及一个免疫细胞化学检测人膀胱癌的方法。该检测方法所检测的抗原为本发明中的人膀胱癌新型标记物AG-CD71，检测抗体为本发明中抗人膀胱癌AG-CD71的单克隆抗体ABC71。
背景技术
膀胱癌是指发生在膀胱黏膜上的恶性肿瘤，是泌尿系统最常见的恶性肿瘤，也是全身十大常见肿瘤之一，占我国泌尿生殖系肿瘤发病率的第一位，在西方其发病率仅次于前列腺癌，居第2位。在我国，由于人口老龄化加重、吸烟人群增多、工业化进程和环境污染加剧，膀胱癌发病率呈逐年上升趋势，总体发病率居男性恶性肿瘤第6位，居泌尿系统肿瘤第1位，男性发病率是女性的4倍，发病率随年龄增长而增加，严重危害着我国人民群众的健康。
目前膀胱癌诊断及随访主要依靠尿道膀胱镜检，但是侵入性的尿道膀胱镜费用昂贵且操作不便，不仅给患者带来较大痛苦，还存在感染、出血等风险。尿液是膀胱癌理想的肿瘤标记物来源。寻找一种N能够通过尿液来检测膀胱癌的高效、简便、快速、灵敏的诊断方法很有必要。
转铁蛋白受体TFRC是细胞表面一种跨膜糖蛋白，分子量约为180kD，由两个同源二聚体亚基通过两条二硫键交联而成。TFRC参与铁的吸收与细胞生长调节，是维持人体正常铁代谢的重要介质。所有正常有核细胞都有低水平的表达，而在高增殖率的细胞中表达水平较高，特别是肿瘤细胞可能由于其对铁的需求增加其表达增加更为显著。近年来，TFRC逐渐成为肿瘤疾病基础和临床研究的一大热点。
因此，基于TFRC的异常糖基化可导致肿瘤细胞恶性程度的改变，探究膀胱癌中TFRC的异常糖基化，为膀胱癌的筛查、诊断、预后判断及治疗提供一种新的靶点，对于膀胱癌的诊断和治疗将具有极其重要的意义。
发明内容
本发明从新鲜的人膀胱癌组织中提取总蛋白免疫小鼠，制备杂交瘤细胞株。用ELISA的方法筛选到一株能与人膀胱癌组织特异性结合的抗体ABC71，该抗体属于IgG1亚类。免疫组织化学证实，ABC71抗体与人膀胱癌组织呈强阳性反应，而与人正常膀胱组织无交叉反应。
本发明通过免疫沉淀结合质谱与糖芯片结果鉴定，ABC71抗体识别的抗原是一种异常糖基化的TFRC，其定位于细胞膜上，其表位为：Fuca1-4(GlcNAcb1-3)[6OSO3]GlcNAc，属于全新的膀胱癌标志物。本发明将ABC71抗体偶联辣根过氧化物酶(HRP)，制备成辣根过氧化物酶标记的抗膀胱癌抗体(ABC71-HRP)，与尿液里脱落的膀胱癌细胞结合后，经DAB显色、苏木素染色涂片，在显微镜下进行镜检。该试剂盒适用于膀胱癌病人的早期筛查、预后监测及病理辅助诊断。
本发明技术方案：
本发明提供了一种全新的人膀胱癌标志物—异常糖基化的转铁蛋白受体TFRC，命名为AG-CD71。并获得了产生抗AG-CD71的单克隆抗体的杂交瘤细胞株，该杂交瘤细胞株分泌单克隆抗体ABC71，单克隆抗体ABC71识别的抗原表位为Fuca1-4(GlcNAcb1-3)[6OSO3]GlcNAc。该单克隆抗体与人膀胱癌组织呈强阳性反应，而与人正常膀胱癌组织无交叉反应。本发明还提供了基于单克隆抗体ABC71的尿脱落细胞免疫细胞化学诊断方法。
具体地，本发明提供了一种异常糖基化的人膀胱癌标志物AG-CD71，其为异常糖基化的转铁蛋白受体TFRC，是在TFRC上包含抗原表位的糖结构Fuca1-4(GlcNAcb1-3)[6OSO3]GlcNAc。所述糖结构Fuca1-4(GlcNAcb1-3)[6OSO3]GlcNAc的结构式为：
Figure GDA0001491442630000031
所述异常糖基化处在所述异常糖基化的转铁蛋白受体TFRC的第104位苏氨酸上。
在一个优选方案中，所述TFRC的氨基酸序列如SEQ ID No:1所示。
本发明的另一个目的是提供针对本发明所述的人膀胱癌标志物AG-CD71的抗体，其能够特异性识别所述作为抗原表位的糖结构Fuca1-4(GlcNAcb1-3)[6OSO3]GlcNAc，所述抗体为多克隆抗体或单克隆抗体，优选单克隆抗体。
本发明的另一个目的是提供一种用于检测人膀胱癌的试剂盒，其包含本发明上述的抗体ABC71。一个优选实施方案中，所述的抗体是抗人膀胱癌AG-CD71的单克隆抗体ABC71，该单克隆抗体由保藏号为CGMCC No.14312的杂交瘤细胞株分泌。
本发明的另一个目的是提供一种缀合物，其中包含所述抗人膀胱癌AG-CD71抗体与选自以下组分组成的物质缀合：生物标记物、抗肿瘤药物、毒素和放射性活性剂。
本发明的另一个目的是提供一种用于检测人膀胱癌的试剂盒，其中包含本发明上述的抗体。
在一个优选实施方案中，在所述的试剂盒中，所述的检测是通过单克隆抗体ABC71偶联辣根过氧化物酶进行，优选将ABC71抗体偶联辣根过氧化物酶，制备成膀胱癌尿脱落细胞检测试剂盒。在一个优选实施方案中，所述待测样品是含人膀胱癌尿脱落细胞。
本发明的另一个目的是提供分泌抗人膀胱癌AG-CD71的单克隆抗体ABC71的杂交瘤细胞株，其保藏号为CGMCC No.14312。
本发明有益技术效果：
(1)筛选并制备了一种抗人膀胱癌的单克隆抗体ABC71，该抗体与人膀胱癌组织呈强阳性反应，而与人正常膀胱组织呈阴性反应；
(2)发现并鉴定了ABC71单克隆抗体识别的抗原表位为Fuca1-4(GlcNAcb1-3)[6OSO3]GlcNAc，属于全新的膀胱癌标志物；
(3)开发了一种基于ABC71抗体的高灵敏度的用于检测人膀胱癌的免疫细胞化学方法。
附图说明
图1为ABC71单抗对人膀胱癌组织切片的免疫组化染色(呈阳性反应)；
图2为ABC71单抗对人正常膀胱组织切片的免疫组化染色(呈阴性反应)；
图3糖芯片检测结果。
具体实施方式
为了使本发明的目的、技术方案及优点更加清楚明白，以下结合附图及实施例，对本发明进行进一步详细描述。应当理解，此处所描述的具体实施例仅仅用于解释本发明，并不用于限定本发明。
相反，本发明涵盖任何由权利要求定义的在本发明的精髓和范围上做的替代、修改、等效方法以及方案。进一步，为了使公众对本发明有更好的了解，在下文对本发明的细节描述中，详尽描述了一些特定的细节部分。对本领域技术人员来说没有这些细节部分的描述也可以完全理解本发明。
实施例1
ABC71单克隆抗体的制备和纯化：
(1)杂交瘤细胞的制备
1)动物免疫及细胞培养：将人膀胱癌组织匀浆后提取总蛋白，免疫Balb/C小鼠(购自北京维通利华实验动物技术有限公司)，按每只小鼠50ug总蛋白的剂量进行腹腔免疫。每隔两周对小鼠再次免疫。小鼠血清效价达到要求后再加强免疫一次，3天后取小鼠脾脏制备成脾细胞悬液，准备进行细胞融合。复苏小鼠骨髓瘤细胞Sp2/0(ATCC CRL-1772)，并用8-AG(8氮杂鸟嘌呤)筛选以维持细胞对HAT的敏感性。
2)细胞融合：将步骤1)制备好的脾细胞悬液与骨髓瘤细胞进行融合，具体方法参照《精编免疫学实验指南》((美国)J.E.科学根(美国)D.H.马古利斯等，科学出版社，2009年1月出版)。将融合后细胞悬液加入含有饲养细胞培养基中培养。24小时后，加HAT选择培养基(购自Sigma公司；HAT即H:Hypoxanthine次黄嘌呤，A:Aminopterin甲氨喋呤，T:Thymidine胸腺嘧啶核苷)进行选择性培养。
3)抗体检测：通过ELISA方法确定分泌抗体的杂交瘤细胞株。
具体方法是：提取膀胱癌组织总蛋白，用0.05mol/L碳酸盐缓冲液(pH9.6)4℃包被过夜，加入5％牛血清白蛋白(BSA)37℃封闭3小时。PBST洗涤3次，然后再加入100ul待检上清，37℃孵育1h。洗涤3次，加入辣根过氧化物酶标记的抗小鼠二抗IgG-HRP(购自北京康为世纪生物科技有限公司)，37℃孵育1h。洗涤3次，加入50ul TMB(购自北京中杉金桥生物技术有限公司)显色5min后，加入50ul终止液。用酶标仪读取波长为450nm的OD值。OD值大于阴性对照OD值的2倍以上的视为阳性。
4)杂交瘤的克隆化和冻存：使用有限稀释法将筛选出的阳性杂交瘤细胞进行克隆化培养。经过5轮的克隆化培养，筛选出高效价单克隆抗体的杂交瘤细胞进行扩大培养。
本发明中获得的一种阳性杂交瘤细胞株，分类命名为：小鼠抗人膀胱癌单克隆抗体杂交瘤细胞株，该杂交瘤细胞株于2017年7月27日保藏于中国微生物菌种保藏管理委员会普通微生物中心(CGMCC，中国，北京)，保藏号为CGMCC No.14312。
(2)ABC71单抗的制备和纯化
将上述分泌单克隆抗体ABC71的杂交瘤细胞株(保藏号CGMCC No.14312)扩大培养，并收集细胞培养上清。采用Protein G对单抗ABC71进行亲和层析纯化。步骤是：首先用磷酸盐缓冲液PBS平衡Protein G亲和层析柱(购自GE公司)；然后将含ABC71单抗的细胞培养上清通过Protein G亲和层析柱；随后用PBS洗涤层析柱，直到流出柱子的洗涤液的OD值接近零；使用0.2mol/L的甘氨酸-HCL溶液(PH2.8)洗脱Protein G亲和层析柱，收集洗脱液，测定OD值。含ABC71单抗的洗脱液经PBS透析后，于-20℃冻存。
实施例2
ABC71单克隆抗体的鉴定：
使用实施例1中制备的ABC71单抗对人膀胱癌组织和人正常膀胱组织切片进行免疫组化，结果如图1、2所示。结果表明，人膀胱癌组织经ABC71单抗免疫组化染色后呈现阳性反应(如图1所示)，而人正常膀胱组织经ABC71单抗免疫组化染色后呈现阴性反应(如图2所示)。
使用实施例1中制备的ABC71单抗，免疫组化检测人膀胱癌组织、人正常膀胱组织，结果如表1所示。结果表明，ABC71抗体与人膀胱癌组织呈强阳性反应，而与人正常膀胱组织无交叉反应。
表1.免疫组化检测抗人膀胱癌单抗ABC71对人膀胱癌组织与正常膀胱组织的免疫反应
组织(癌组织和正常组织) ABC71抗体
人膀胱癌组织(病人#1) 强阳性(+++)
人正常膀胱组织(病人#1) 阴性(－)
人膀胱癌组织(病人#2) 强阳性(+++)
人正常膀胱组织(病人#2) 阴性(－)
人膀胱癌组织(病人#3) 强阳性(+++)
人正常膀胱组织(病人#3) 阴性(－)
人膀胱癌组织(病人#4) 阳性(++)
人正常膀胱组织(病人#4) 阴性(－)
人膀胱癌组织(病人#5) 强阳性(+++)
人正常膀胱组织(病人#5) 阴性(－)
实施例3
AG-CD71抗原的制备：
1)总蛋白提取：100mg人膀胱癌组织，研磨成匀浆后，加入2ml的三去污裂解液，4℃裂解10分钟，12000rpm离心20分钟，取上清，即为人膀胱癌组织总蛋白。
2)免疫沉淀：加入50ugABC71单抗，4℃孵育2h。
3)抗原鉴定：加入50ul Protein G beads，4℃孵育2h。用PBS洗涤beads，然后用0.2mol/L的甘氨酸-HCL溶液(PH2.8)洗脱beads，进行质谱分析。质谱结果如表2所示。质谱结果表明，ABC71的抗原为转铁蛋白受体TFRC。经过分析ABC71单抗的糖芯片数据(如图3所示)发现，ABC71单抗识别的抗原表位为TFRC的糖链，该表位是Fuca1-4(GlcNAcb1-3)[6OSO3]GlcNAc。由于ABC71单抗只特异性的识别人膀胱癌组织，表明该抗原为异常糖基化的TFRC，其表位Fuca1-4(GlcNAcb1-3)[6OSO3]GlcNAc只表达于人膀胱癌组织细胞。
表2.ABC71抗原的质谱鉴定
Accession Mass Score
TFRC HUMAN 186395 197
Tubulin HUMAN 36483 53
Filaggrin HUMAN 19176 42
Dermcidin HUMAN 25320 35
Actin,cytoplasmic 1 HUMAN 36127 32
Collagen alpha-2HUMAN 27329 30
Protein NDRG1 HUMAN 63517 30
Tyrosine-protein kinase HUMAN 35379 25
Zinc finger protein 517 HUMAN 24258 20
Stress-70 protein HUMAN 13576 20
实施例4
膀胱癌尿脱落细胞检测试剂盒：
利用本发明中实施例1中制备的ABC71单抗，将ABC71抗体偶联辣根过氧化物酶，制备成辣根过氧化物酶标记的ABC71单抗(ABC71-HRP)。收集尿脱落细胞，加入ABC71-HRP，经DAB染色、苏木素复染，最后在显微镜下进行镜检。该试剂盒适用于肿瘤病人的早期筛查、预后监测及病理辅助诊断。实验证实本发明的人膀胱癌尿脱落细胞检测试剂盒与已有技术相比，具有以下积极效果：(1)灵敏度高，由于ABC71单抗直接结合在膀胱癌细胞膜表面，偶联ABC71单抗的HRP催化底物DAB显色，该方法的灵敏度远高于常规的脱落细胞学的检测方法；(2)特异性强，由于该方法采用了特异性结合膀胱癌细胞的ABC71单抗，它能特异性的识别膀胱癌细胞；(3)方便快捷，成本低廉，节省患者就医费用，由于该方法灵敏度高、特异性强等特点，对膀胱癌患者的检出率高，避免重复检测所造成的浪费。
具体实验方法如下：
1)尿脱落细胞采集：膀胱癌患者的尿液来自北京大学第一医院。
2)尿脱落细胞免疫细胞化学染色：取10ml新鲜尿液，加入100ul ABC71-HRP，室温孵育10分钟。1000rpm离心5分钟，弃上清，PBS洗涤2次，加入DAB显色液室温显色10分钟，PBS洗涤，苏木素复染30秒，PBS再次洗涤后涂片于载玻片用于镜检。
3)镜检诊断：根据膀胱癌细胞的形态特征，判断病理染色结果。
ABC71-HRP法与常规涂片法阳性检出率比较结果，如表3所示。结果表明，采用ABC71-HRP检测了来自北京大学第一医院57例膀胱癌患者的尿液，ABC71-HRP的阳性检出率为91.23％(52/57)，远高于常规脱落细胞涂片的阳性检出率40.35％(23/57)。因此，膀胱癌细胞特异表达的AG-CD71是一个很有应用前景的膀胱癌标志物，抗膀胱癌AG-CD71的单克隆抗体ABC71能够高效检测膀胱癌患者尿液中的肿瘤细胞。
表3.ABC71-HRP法与常规涂片法阳性检出率比较
Figure GDA0001491442630000111
Figure GDA0001491442630000121
“+”表示检出膀胱癌细胞；“－”表示未检出膀胱癌细胞。
SEQUENCE LISTING
<110> 李翀
<120> 人膀胱癌标志物AG-CD71及其抗体ABC71和应用
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<170> PatentIn version 3.5
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Claims (9)

1.一种人膀胱癌标志物AG-CD71,其特征在于,其为异常糖基化的转铁蛋白受体TFRC,所述异常糖基化的转铁蛋白受体TFRC是指TFRC上带有作为抗原表位的糖结构Fuca1-4(GlcNAcb1-3)[6OS03]G1cNAc，所述异常糖基化处在所述异常糖基化的转铁蛋白受体TFRC的第104位苏氨酸上；
所述糖结构Fuca1-4(GlcNAcb1-3)[60S03]G1cNAc的结构式为:
Figure FDA0002508532880000011
2.根据权利要求1所述人膀胱癌标志物AG-CD71,其特征在于,所述异常糖基化的转铁蛋白受体TFRC的氨基酸序列如SEQ ID No:1所示。
3.针对根据权利要求1或2所述的人膀胱癌标志物AG-CD71的抗体,其特征在于,所述抗体能够特异性识别所述作为抗原表位的糖结构Fuca1-4(GlcNAcb1-3)[6OS03]GlcNAc，所述抗体为特异性抗人膀胱癌AG-CD71的单克隆抗体ABC71,该单克隆抗体由保藏号为CGMCCNo.14312的杂交瘤细胞株分泌。
4.根据权利要求3所述抗体在制备膀胱癌诊断试剂中的应用。
5.一种缀合物,其特征在于,抗人膀胱癌标志物AG-CD71的抗体ABC71与选自以下组分组成的物质缀合：生物标记物、抗肿瘤药物、毒素、放射性活性剂、磁颗粒；所述抗体ABC71为单克隆抗体，由保藏号为CGMCCNo.14312的杂交瘤细胞株分泌。
6.一种用于检测或治疗人膀胱癌的试剂盒,其特征在于,所述试剂盒包括权利要求3所述抗体。
7.根据权利要求6所述试剂盒,其特征在于,通过免疫细胞化学进行检测，所述免疫细胞化学是将权利要求3所述抗体偶联辣根过氧化物酶,制备成膀胱癌尿脱落细胞检测试剂盒。
8.根据权利要求6所述试剂盒,所述试剂盒的检测样品为含膀胱癌脱落细胞的人尿液。
9.分泌抗人膀胱癌转铁蛋白受体TFRC的单克隆抗体ABC71的杂交瘤细胞株,于2017.07.27日保藏于中国微生物菌种保藏管理委员会普通微生物中心,保藏号为CGMCCNO.14312。
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