CN117264067A - 一种akr1c1免疫组织化学抗体及其应用 - Google Patents
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Abstract
本发明公开了一种AKR1C1免疫组织化学抗体及其应用。本发明的AKR1C1免疫组织化学抗体,包括重链可变区和轻链可变区,重链可变区的氨基酸序列如SEQ ID NO:2所示;轻链可变区的氨基酸序列如SEQ ID NO:4所示;或者,重链可变区的氨基酸序列如SEQ ID NO:6所示;轻链可变区的氨基酸序列如SEQ ID NO:8所示。本发明的抗体可用于鉴别与AKR1C1相关的正常细胞和肿瘤细胞,以及乳腺癌各亚型的鉴别,和包括脂肪肉瘤、平滑肌肉瘤、胃肠间质瘤和横纹肌肉瘤在内的组织肿瘤亚型的鉴别,是一种极具应用潜力的免疫组化检测用抗体。
Description
技术领域
本发明涉及一种AKR1C1免疫组织化学抗体及其应用,属于生物医药技术领域。
背景技术
免疫组织化学是利用抗原与抗体间的特异性结合原理和特殊的标记技术,对组织和细胞内的特定抗原或抗体进行定位、定性或定量检测的技术。免疫组织化学技术具有特异性强、敏感性高、定位准确、形态与功能相结合等特点,有利于病理学领域的深入研究,在现代病理诊断中起重要作用。
病理科日常工作使用的免疫组织化学抗体品种达数百种,对人类疾病的诊断和治疗发挥着重要的作用。随着人类对疾病认识的不断提高,病理科应用的抗体种类也逐渐增加。在近十年人类基因组计划完成和蛋白质谱计划推进的过程中,越来越多抗体的潜在价值等待被发掘,等待不断扩展和添加到病理科抗体应用的目录中。
免疫组织化学中所使用的抗体,又称为一抗(Primary Antibody),根据其制备方式可分为单克隆抗体和多克隆抗体。单克隆抗体因其特异性高广受免疫组化使用者的青睐,在病理科使用的抗体中占绝大多数。
免疫组织化学抗体的应用价值包括多个方面:首先,免疫组织化学抗体能够辅助诊断和鉴别诊断,为病理诊断和鉴别诊断提供客观证据。对于感染性疾病,通过免疫组织化学技术识别特定的细菌、病毒等微生物,提高感染性疾病病理诊断的准确性。对于肿瘤性疾病,免疫组织化学也通过多种途径来提供客观的诊断依据辅助病理诊断工作:如肿瘤良恶性判断;肿瘤细胞来源/分化方向;帮助病理医生判断原发性还是转移性;鉴别诊断肿瘤类型;肿瘤分级;肿瘤微小转移灶的发现和确定等。其次,为疾病治疗提供客观的指导方案,即伴随诊断:分子靶向药物肿瘤靶标的测定;肿瘤化学性药物治疗反应的预测;肿瘤预后的判断等。正是由于免疫组织化学在临床上广谱的应用价值,因此,抗体的应用研究方兴未艾。
中国专利CN 114874318 A公开了一种NG-2免疫组织化学抗体,主要应用于与NG-2相关的正常细胞与肿瘤细胞的识别,以及食管鳞状细胞癌和食管基底样鳞状细胞癌的鉴别诊断;中国专利CN116217721A公开了一种C14orf78免疫组织化学抗体及其制备方法与应用,所述的C14orf78免疫组织化学抗体主要应用于与C14orf78相关的正常细胞与肿瘤细胞的识别,以及甲状腺肿瘤、肝脏肿瘤和肾脏肿瘤的鉴别诊断。
但是目前还没有关于AKR1C1基因和蛋白相关的免疫组化抗体的研究和报道。
发明内容
本发明所要解决的技术问题是:AKR1C1免疫组织化学抗体如何制备和如何应用的技术问题。
为了解决上述技术问题,本发明采取了以下技术方案:
本发明的第一方面,提供一种AKR1C1免疫组织化学抗体,所述抗体包括重链可变区和轻链可变区,重链可变区的氨基酸序列如SEQ ID NO:2所示;轻链可变区的氨基酸序列如SEQ ID NO:4所示;或者,重链可变区的氨基酸序列如SEQ ID NO:6所示;轻链可变区的氨基酸序列如SEQ ID NO:8所示。
本发明的第二方面,提供了一种AKR1C1免疫组织化学抗体的重链可变区和轻链可变区的编码基因,所述重链可变区的编码基因序列如SEQ ID NO:1所示,所述轻链可变区的编码基因序列如SEQ ID NO:3所示;或者,所述重链可变区的编码基因序列如SEQ ID NO:5所示,所述轻链可变区的编码基因序列如SEQ ID NO:7所示。
本发明的第三方面,提供了一种AKR1C1免疫组织化学抗体在制备鉴别与AKR1C1相关的正常细胞和肿瘤细胞的试剂盒中的应用,所述抗体包括重链可变区和轻链可变区,重链可变区的氨基酸序列如SEQ ID NO:2所示,轻链可变区的氨基酸序列如SEQ ID NO:4所示;或者,重链可变区的氨基酸序列如SEQ ID NO:6所示;轻链可变区的氨基酸序列如SEQID NO:8所示。
本发明的第四方面,提供了一种AKR1C1免疫组织化学抗体在制备鉴别乳腺癌亚型的诊断试剂盒中的应用,所述抗体包括重链可变区和轻链可变区,重链可变区的氨基酸序列如SEQ ID NO:2所示,轻链可变区的氨基酸序列如SEQ ID NO:4所示;或者,重链可变区的氨基酸序列如SEQ ID NO:6所示;轻链可变区的氨基酸序列如SEQ ID NO:8所示。
优选地,所述乳腺癌亚型包括乳腺大汗腺癌、乳腺浸润性导管癌、乳腺非特殊浸润性癌、乳腺黏液癌、乳腺实性乳头状癌、乳腺微乳头状癌、乳腺小叶癌、神经内分泌癌、鳞状细胞癌、纤维腺瘤和分叶状肿瘤中的任意一种。
本发明的第五方面,提供了一种AKR1C1免疫组织化学抗体在制备鉴别组织肿瘤亚型的诊断试剂盒中的应用,所述组织肿瘤亚型包括脂肪肉瘤、平滑肌肉瘤、胃肠间质瘤和横纹肌肉瘤中的任意一种;所述抗体包括重链可变区和轻链可变区,重链可变区的氨基酸序列如SEQ ID NO:2所示,轻链可变区的氨基酸序列如SEQ ID NO:4所示;或者,重链可变区的氨基酸序列如SEQ ID NO:6所示;轻链可变区的氨基酸序列如SEQ ID NO:8所示。
本发明从发现的候选分子库中,制备一种AKR1C1免疫组织化学抗体,其能够特异性识别大汗腺和乳腺的大汗腺癌,能够鉴别乳腺大汗腺和浸润性导管癌以及其他少见类型的乳腺癌,正常大汗腺组织阳性率98%,正常乳腺导管和腺泡为阴性,乳腺大汗腺癌阳性率90%,且多为弥漫强阳性,乳腺其他浸润导管癌阳性率约15%,往往为局灶阳性,特殊类型乳腺癌,如乳腺黏液癌、实性乳头状癌、微乳头状癌、小叶癌为阴性;此外,本发明的AKR1C1免疫组织化学抗体还可以鉴别脂肪肉瘤和平滑肌肉瘤、横纹肌肉瘤以及胃肠间质瘤。因此,本发明的AKR1C1免疫组织化学抗体是一种极具潜力的各种亚型乳腺癌和组织肿瘤亚型的鉴别诊断指标,并具有进一步探索作为其他器官、系统、组织和细胞水平潜在的鉴别、预测和靶标的价值。
本发明与现有技术相比,具有如下有益效果:
(1)本发明的AKR1C1免疫组织化学抗体,为在组织和细胞上进行免疫组织/细胞化学染色的抗体,用于将AKR1C1相关的正常细胞和肿瘤细胞显示出来,达到组织和细胞分型以及肿瘤组织鉴别诊断的目的;此为,基于本发明提供的方案,还可以为探索AKR1C1进一步作为其他器官、系统、组织和细胞水平潜在的鉴别、预测和靶标的应用提供方向;
(2)本发明的AKR1C1免疫组织化学抗体,能够特异性识别大汗腺和乳腺的大汗腺癌,能够鉴别乳腺大汗腺和浸润性导管癌以及其他少见类型的乳腺癌,正常大汗腺组织阳性率98%,正常乳腺导管和腺泡为阴性,乳腺大汗腺癌阳性率90%;此外,本发明的AKR1C1免疫组织化学抗体还可以鉴别脂肪肉瘤和平滑肌肉瘤、横纹肌肉瘤以及胃肠间质瘤;极具应用潜力。
(3)乳腺肿瘤类型丰富,包括浸润性导管癌、浸润性小叶癌、大汗腺癌、化生性癌、实性乳头状癌、黏液癌、神经内分泌癌、鳞状细胞癌,以及纤维腺瘤、分叶状肿瘤等,通过免疫组化检测可以得到AKR1C1在乳腺各亚型表达的翔实数据,最终有效地将AKR1C1用于乳腺大汗腺癌的识别以及各亚型的鉴别中。
附图说明
图1:在正常组织中免疫组织化学抗体AKR1C1-7B11A10的免疫组化检测的染色图;
图2:在乳腺肿瘤组织中免疫组织化学抗体AKR1C1-7B11A10的免疫组化检测染色图。
图3:在脂肪肉瘤、平滑肌肉瘤、胃肠间质瘤、横纹肌肉瘤中免疫组织化学抗体AKR1C1-7B11A10的免疫组化检测染色图。
具体实施方式
为使本发明更明显易懂,兹以优选实施例,并配合附图作详细说明如下。
下述实施例中所使用的试验方法如无特殊说明,均为常规方法;所使用的材料、试剂等,如无特殊说明,为可从商业途径得到的试剂和材料。
实施例1:单克隆抗体的制备
1、抗原准备
1.1在数据库中查找目的基因序列并合成,构建到原核表达载体;
1.2将质粒转入大肠杆菌,用IPTG诱导蛋白表达;
1.3用亲和纯化的方法提纯蛋白。
2、动物免疫与效价测定
2.1将抗原与佐剂混合,在乳化器上进行乳化,将乳化好的抗原通过腹腔注射进入小鼠体内,一共免疫5只小鼠,间隔3周后注射第二次,间隔2周后注射第三次。
2.2第三次免疫后10天取小鼠尾部血后分离血清,通过间接ELISA法测定小鼠血清效价。
2.3选取血清效价最好的一只小鼠,融合前三天进行加强免疫,不加佐剂,只注射抗原。
3、细胞融合
3.1提前复苏并扩大培养SP2/0细胞;
3.2处死小鼠,取其脾脏并研磨制成细胞悬液,将骨髓瘤细胞与脾细胞按比例混合后,加入PEG促进融合。融合好的细胞移至96孔板中用HAT培养基培养,一段时间后,未融合成功的细胞死亡。
4、细胞筛选
4.1取细胞上清液,采用ELISA+免疫组化双重筛选,将效价高、定位清晰的孔转移至24孔板置37℃CO2培养箱中扩大培养。
4.2有限稀释法进行克隆化培养:将24孔板细胞制成悬液移入新96孔板中,尽量保证每孔只含一个细胞,置37℃CO2培养箱中培养。10天后采用ELISA+免疫组化双重筛选,挑选效价高、定位清晰的克隆扩培。
5、亚型鉴定和抗体测序
5.1采用ELISA法分别对挑选的效价高、定位清晰克隆的轻链和重链的亚型进行鉴定。选择最好的两个阳性亚克隆进行腹水生产准备,阳性亚克隆代号为:7B11A10和5C5G5。提供7B11A10阳性亚克隆细胞株并进行下一步的腹水生产和纯化,纯化抗体量为10mg/克隆。
5.2采用RACE方法,对杂交瘤细胞重链和轻链分别进行测序,具体步骤为:杂交瘤细胞的培养;杂交瘤细胞mRNA提取;RNA反转录获得cDNA;cDNA PCR获得抗体重链/轻链可变区基因;抗体基因连接至T载体,挑选阳性克隆并测序;生物信息学分析测序结果,得到抗体序列,具体测序结果如下:
7B11A10克隆:
重链可变区基因序列(Heavy chain variable region sequence,SEQ ID NO:1)
H-:
CAGGTGCAGCTGCAGCAGTCTGGGGCTGAGCTTGTGAGGCCAGGGGCCTTAGTCAAGTTGTCCTGCAAAGCTTCTGGCTTCAACATTAAAGACTACTAT ATTCACTGGGTGAAGCAGAGGCCTGAACAGGGCCTGGAGTGGATTGGATGGATTGATCCTGAGAATGGTCATACTGTATATGACCCGAAGTTCCAGGGCAAGGCCATTATAACAGCAGACACATCCTCCAACACAGCCTACCTGCAACTCAGCAGCCTGACATCTGAGGACACTGCCGTCTATTACTGTGCTAGCCCTTATGCTTACGACGGATTACTAGCTATGGACCACTGGGGTCAAGGAACCTCAGTCACCGTCTCCTCA;
重链可变区氨基酸序列(Translated protein,SEQ ID NO:2):
QVQLQQSGAELVRPGALVKLSCKASGFNIKDYYIHWVKQRPEQGLEWIG WIDPENGHTVYDPKFQGKAIITADTSSNTAYLQLSSLTSEDTAVYYCASPYAYD GLLAMDHWGQGTSVTVSS;
轻链可变区基因序列(Light chain variable region sequence,SEQ ID NO:3)
K-:
GATATTGTGATGACTCAGACTGCACCCTCTGTTCCTGTCACTCCTGGAGAGTCAGTATCCATCTCCTGCAGGTCTAGTAAGAGTCTCCTGCATAGTACTGGCAATACTTACTTGTATTGGTTCCTGCAGAGGCCAGGCCAGTCTCCTCAGCTCCTGATATATCGGATGTCCAACCTTGTCTCAGGAGTCCCAGACAGGTTCAGTGGCAGTGGGTCAGGAACTGCTTTCACACTGACAATCAGTAGAGTGGAGGCTGAGGATGTGGGTGTTTATTACTGTATGCAACATCTAGAATATCCTTTCACGTTCGGTGCTGGGACCAAGCTGGAGCTGAAACGG;
轻链可变区氨基酸序列(Translated protein,SEQ ID NO:4)
DIVMTQTAPSVPVTPGESVSISCRSSKSLLHSTGNTYLYWFLQRPGQSPQL LIYRMSNLVSGVPDRFSGSGSGTAFTLTISRVEAEDVGVYYCMQHLEYPFTFG AGTKLELKR。
5C5G5克隆:
重链可变区基因序列(Heavy chain variable region sequence,SEQ ID NO:5)
H-:
CAAGTACAGCTGCAGGAGTCTGGAGGAGGCTTGGTGCAACCAGGAGGATCCATGAAACTCTCCTGTGTTGCCTCTGGATTCATTGTCAGTAACTATTGGATGAACTGGGTCCGCCAGTCTCCAGAGAAGGGGCTTGAGTGGGTTGCTGAAATTAGATTGAAATCTAATAATTATGCAACACATTATGCGGAGTCTGTGAAA GGGAGGTTCACCATCTCAAGAGATGATTCCAAAAGTAGTGTCTATCTGCAAATGAACAACTTAAGAGTTGAAGACACTGGCATTTATTACTGTACCATCATTAGTTATTGGGGCCAAGGCACCACTCTCACAGTCTCCTCA;
重链可变区氨基酸序列(Translated protein,SEQ ID NO:6)
QVQLQESGGGLVQPGGSMKLSCVASGFIVSNYWMNWVRQSPEKGLEWV AEIRLKSNNYATHYAESVKGRFTISRDDSKSSVYLQMNNLRVEDTGIYYCTIIS YWGQGTTLTVSS;
轻链可变区基因序列(Light chain variable region sequence,SEQ ID NO:7)
K-:
GATATTGTGATGACGCAGGCTGCATTCTCCAATCCAGTCACTCTTGGAACATCAGCTTCCATCTCCTGCAGGTCTAGTAAGAGTCTCCTACATAGTAATGGCATCACTTATTTGTATTGGTATCTGCAGAAGCCAGGCCAGTCTCCTCAGCTCCTGATTTATCAGATGTCCAACCTTGCCCCAGGAGTCCCAGACAGGTTCAGTAGCAGTGGGTCAGGAACTGATTTCACACTGAGAATCAGCAGAGTGGAGGCTGAGGATGTGGGTGTTTATTACTGTGGTCAAAATCTAGAACTTCCGTGGACGTTCGGTGGAGGCACGAAGCTGGAAATCAAACGG;
轻链可变区氨基酸序列(Translated protein,SEQ ID NO:8)
DIVMTQAAFSNPVTLGTSASISCRSSKSLLHSNGITYLYWYLQKPGQSPQL LIYQMSNLAPGVPDRFSSSGSGTDFTLRISRVEAEDVGVYYCGQNLELPWTFG GGTKLEIKR。
6、单克隆抗体生产
6.1动物体内诱生法制备腹水:挑选10周龄的健康BALB/C小鼠,提前3-7天腹腔注射不完全佐剂0.5ml。将挑选的效价高、定位清晰的克隆7B11A10进行培养,收集2×106细胞用无菌PBS清洗后,注射到小鼠腹腔内,一般7-10左右腹水产生较充分,可收获腹水。
6.2抗体纯化:腹水离心,收集上清,采用Protein A/G亲和纯化的方法从上清中提纯抗体并做ELISA+免疫组化双重验证。
实施例2
用AKR1C1-7B11A10抗体在皮肤和器官等正常组织芯片上进行免疫组化检测:
1、选取含有皮肤、睾丸、前列腺、输尿管、扁桃体、甲状腺、阑尾、胆囊、腹腔软组织、脾脏、肾脏组织、骨髓、肌腱、脑组织、肾上腺、肺、胎盘、胰腺、颌下腺、腮腺、胃黏膜、小肠黏膜、十二指肠黏膜、肝组织等正常组织的组织芯片。
2、PH9.0抗原修复。
3、在罗氏平台上进行AKR1C1-7B11A10抗体全自动免疫组化检测。
4、结果发现AKR1C1-7B11A10在皮肤、睾丸、前列腺、扁桃体、脾脏、神经束、脑组织等不表达,在肺、胎盘、颌下腺、卵巢、腮腺等也不表达。而输尿管上皮强表达,有器官特异性,如图1所示。
实施例3
用AKR1C1-7B11A10抗体在乳腺肿瘤组织芯片上进行免疫组化检测:
1、选取含不同组织类型的乳腺肿瘤的组织芯片。
2、PH9.0抗原修复。
3、在罗氏平台上进行AKR1C1-7B11A10全自动免疫组化检测。
4、结果在乳腺大汗腺癌中AKR1C1-7B11A10强阳性,阳性率86%,在浸润性癌和其他少见亚型的乳腺癌中为阴性,在乳腺癌亚型中有鉴别诊断价值,如图2所示。
实施例4
用AKR1C1-7B11A10抗体在脂肪肉瘤和其他部分软组织肿瘤组织芯片上进行免疫组化检测:
1、选取含脂肪肉瘤、平滑肌肉瘤、胃肠间质瘤、横纹肌肉瘤的组织芯片。
2、PH9.0抗原修复。
3、在罗氏平台上进行AKR1C1-7B11A10全自动免疫组化检测。
4、结果在脂肪肉瘤中,AKR1C1-7B11A10强阳性,阳性率90%,在平滑肌肉瘤、胃肠间质瘤、横纹肌肉瘤为阴性,有鉴别诊断价值,如图3所示。
Claims (6)
1.一种AKR1C1免疫组织化学抗体,其特征在于,所述抗体包括重链可变区和轻链可变区,重链可变区的氨基酸序列如SEQ ID NO:2所示;轻链可变区的氨基酸序列如SEQ ID NO:4所示;或者,重链可变区的氨基酸序列如SEQ ID NO:6所示;轻链可变区的氨基酸序列如SEQ ID NO:8所示。
2.AKR1C1免疫组织化学抗体的重链可变区和轻链可变区的编码基因,其特征在于,所述重链可变区的编码基因序列如SEQ ID NO:1所示,所述轻链可变区的编码基因序列如SEQID NO:3所示;或者,所述重链可变区的编码基因序列如SEQ ID NO:5所示,所述轻链可变区的编码基因序列如SEQ ID NO:7所示。
3.一种AKR1C1免疫组织化学抗体在制备鉴别与AKR1C1相关的正常细胞和肿瘤细胞的试剂盒中的应用,其特征在于,所述抗体包括重链可变区和轻链可变区,重链可变区的氨基酸序列如SEQ ID NO:2所示,轻链可变区的氨基酸序列如SEQ ID NO:4所示;或者,重链可变区的氨基酸序列如SEQ ID NO:6所示;轻链可变区的氨基酸序列如SEQ ID NO:8所示。
4.一种AKR1C1免疫组织化学抗体在制备鉴别乳腺癌亚型的诊断试剂盒中的应用,其特征在于,所述抗体包括重链可变区和轻链可变区,重链可变区的氨基酸序列如SEQ ID NO:2所示,轻链可变区的氨基酸序列如SEQ ID NO:4所示;或者,重链可变区的氨基酸序列如SEQID NO:6所示;轻链可变区的氨基酸序列如SEQ ID NO:8所示。
5.如权利要求4所述的应用,其特征在于,所述乳腺癌亚型包括乳腺大汗腺癌、乳腺浸润性导管癌、乳腺非特殊浸润性癌、乳腺黏液癌、乳腺实性乳头状癌、乳腺微乳头状癌、乳腺小叶癌、神经内分泌癌、鳞状细胞癌、纤维腺瘤和分叶状肿瘤中的任意一种。
6.一种AKR1C1免疫组织化学抗体在制备鉴别组织肿瘤亚型的诊断试剂盒中的应用,其特征在于,所述组织肿瘤亚型包括脂肪肉瘤、平滑肌肉瘤、胃肠间质瘤和横纹肌肉瘤中的任意一种;所述抗体包括重链可变区和轻链可变区,重链可变区的氨基酸序列如SEQ ID NO:2所示,轻链可变区的氨基酸序列如SEQ ID NO:4所示;或者,重链可变区的氨基酸序列如SEQID NO:6所示;轻链可变区的氨基酸序列如SEQ ID NO:8所示。
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Application publication date: 20231222 Assignee: FUZHOU MAIXIN BIOTECH, Co.,Ltd. Assignor: ZHONGSHAN HOSPITAL, FUDAN University Contract record no.: X2024980001345 Denomination of invention: An AKR1C1 immunohistochemical antibody and its application License type: Common License Record date: 20240124 |