CN116425873B - 一种抗ck6重组兔单克隆抗体及其应用 - Google Patents
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Abstract
本发明涉及一种抗CK6重组兔单克隆抗体及其应用,所述抗CK6重组兔单克隆抗体包括重链可变区和轻链可变区,所述重链可变区的氨基酸序列如SEQ ID No.4所示;所述轻链可变区的氨基酸序列如SEQ ID No.5所示。相比市售的抗CK6单克隆抗体,本发明提供的抗CK6重组兔单克隆抗体,与CK6蛋白具有更高亲和力,可高特异性和高灵敏度地识别和检测肿瘤细胞或免疫细胞上CK6蛋白的表达,可应用于免疫组织化学(IHC)、间接ELISA、免疫印记(Western blotting)、抗体芯片制备、流式细胞术等检测与筛查领域,有利于获得更准确的检测和评估结果,并降低检测成本和背景信号的干扰。
Description
技术领域
本发明属于免疫化学技术领域,尤其涉及一种抗CK6重组兔单克隆抗体及其应用,特别是在免疫组织化学检测方面的应用。
背景技术
细胞角蛋白6(Cytokeratin 6, CK6),分子量约为56 kDa,通常与细胞角蛋白16或细胞角蛋白17(Cytokeratin 16&17, CK16&CK17)成对出现,属于II型碱性细胞角蛋白。在正常组织中,CK6可标记鳞状上皮和导管上皮的基底细胞以及部分鳞状上皮发生层细胞、肌上皮细胞和间皮细胞,腺上皮细胞不表达。因此,可用于鳞癌和腺癌、间皮瘤和腺癌的鉴别诊断。亦可用于导管上皮良、恶性增生的鉴别诊断。CK6通常与CK5密切相关,它们具有相似的组织分布,并且在许多非角化复层鳞状上皮中以不同比例呈现,如舌粘膜、气管基底上皮、表皮基底细胞、毛囊、皮肤皮脂腺和汗腺、乳腺腔细胞、前列腺基底细胞、尿道上皮细胞、阴道和宫颈内膜粘膜等。因此筛选一株高灵敏度、强特异性的抗CK6抗体,对识别和检测肿瘤细胞或免疫细胞上CK6蛋白的表达具有非常重要的作用。
发明内容
(一)要解决的技术问题
鉴于现有技术的上述缺点、不足,本发明提供一种抗CK6重组兔单克隆抗体及其应用,所述抗CK6重组兔单克隆抗体应用广泛,并能准确识别CK6的表达,经过多种不同组织的免疫组织化学检测发现,该抗体可以很好的检测到肿瘤细胞或免疫细胞上CK6蛋白的表达,可应用于免疫组织化学(IHC)、间接ELISA、免疫印记(Western blotting)、抗体芯片制备、流式细胞术等检测与筛查领域。本发明还涉及编码该抗CK6重组兔单克隆抗体的核苷酸序列、重组质粒或表达载体、制备方法及抗CK6重组兔单克隆抗体在CK6蛋白检测方法或装置中的应用等。
(二)技术方案
为了达到上述目的,本发明采用的主要技术方案包括:
第一方面,本发明提供一种抗CK6重组兔单克隆抗体,其包括重链可变区和轻链可变区,所述重链可变区的氨基酸序列如SEQ ID No.4所示;所述轻链可变区的氨基酸序列如SEQ ID No.5所示。
抗CK6重组兔单克隆抗体(CK6兔源抗体)可用于免疫组织化学检测,能高特异性和高灵敏度地识别和检测肿瘤细胞或免疫细胞上CK6蛋白的表达。
所述抗CK6单克隆抗体由哺乳动物细胞重组表达获得。具体地,本发明提供的抗CK6重组兔单克隆抗体是通过兔杂交瘤融合筛选,293细胞真核表达产生。在制备所述抗CK6单克隆抗体时,免疫兔子(新西兰大白兔)用的抗原为合成多肽,所述合成多肽的氨基酸序列如SEQ ID No.1所示,其由人工化学合成得到。免疫兔子后,经细胞融合、克隆筛选,获得可高效分泌单克隆抗体的阳性杂交瘤细胞系,并使用分子克隆技术获得编码所述抗体的重链氨基酸序列和轻链氨基酸序列的核苷酸序列,将核苷酸序列构建在真核表达载体上,通过转染试剂转染到293细胞系,收集细胞上清,经Protein A柱亲和层析纯化细胞上清,获得兔单克隆抗体。免疫组织化学检测显示该抗体能特异性识别CK6蛋白。
所述抗CK6单克隆抗体能够识别重组CK6抗原蛋白和肿瘤细胞和免疫细胞上的CK6分子;所述抗CK6单克隆抗体还可以应用在免疫组化病理诊断剂中。
第二方面,本发明提供编码基因,用于编码上述抗CK6重组兔单克隆抗体。
优选地,所述编码基因包括如SEQ ID No.2所示的DNA序列,用于编码所述抗CK6重组兔单克隆抗体的重链可变区;以及如SEQ ID No.3所示的DNA序列用于编码所述抗CK6重组兔单克隆抗体的轻链可变区。
第三方面,本发明涉及一种核酸分子,其包括用于编码所述抗CK6重组兔单克隆抗体的编码基因。
第四方面,本发明保护一种表达载体或重组质粒,其包括上述的核酸分子。
第五方面,本发明提供一种抗CK6重组兔单克隆抗体的制备方法,采用上述表达载体转染细胞,培养转染后的细胞,收集细胞上清液并纯化,得到所述抗CK6重组兔单克隆抗体。
更优选地,所述制备方法包括如下步骤:
(1)免疫兔子:先对CK6蛋白分子序列进行分析,依据CK6在细胞膜上的结构、抗原性、组成氨基酸的亲疏水性以及二级结构,选择和使用合适的多肽序列作为免疫原,经由KLH或OVA偶联后作为免疫原,免疫兔子;所述多肽为SEQ ID No.1所示的人工合成多肽;
(2)制备杂交瘤细胞系:经细胞融合、克隆筛选,获得可高效分泌抗体的阳性杂交瘤稳定细胞系,从杂交瘤细胞系中分离出总RNA;
(3)获得抗体序列:利用特异性的引物,通过PCR扩增技术获得抗体重链可变区和抗体轻链可变区的核苷酸序列;
(4)抗体表达和纯化:将所述核苷酸序列克隆至表达载体中,使用转染方法瞬时转染培养的细胞,培养后收集上清,使用Protein A纯化上清液,得到纯度>95%的抗体。
第六方面,所述的抗CK6重组兔单克隆抗体、编码基因、核酸分子、表达载体或重组质粒在制备CK6蛋白分子检测装置中的应用。所述检测装置包括但不限于CK6检测试剂盒、抗体芯片或抗体探针等。第七方面,本发明还提供一种CK6检测试剂盒,其包括上述的抗CK6重组兔单克隆抗体和免疫组织化学检测试剂。
优选地,所述CK6检测试剂盒包括:抗CK6重组兔单克隆抗体、HRP酶标二抗、EDTA修复液、过氧化氢酶封闭液、DAB浓缩液、DAB缓冲液、苏木素、返蓝液。
在进行免疫组织化检测时,检测步骤包括脱蜡、抗原修复、内源性过氧化物酶失活、封闭、一抗孵育、二抗孵育、DAB显色、复染、脱水、封片和镜检等。
(三)有益效果
本发明提供的抗CK6重组兔单克隆抗体,与CK6蛋白分子的结合具有高特异性和高灵敏度,能够特异性地识别和检测肿瘤细胞或免疫细胞上CK6蛋白的表达,在检测CK6蛋白时呈阳性高表达,因此该抗体可应用于免疫组织化学(IHC)、间接ELISA、免疫印记(Westernblotting)、抗体芯片制备、流式细胞术等检测与筛查领域,有利于获得准确的评估和检测结果。本发明的564G2D3克隆的CK6重组兔单克隆抗体由于其特异性好,阳性信号强等特点,在IHC染色中评分更容易,对于检测区分癌症更准确。
附图说明
图1为本发明制备的抗CK6单克隆抗体和市售抗体在人肺鳞癌组织的免疫组化检测结果图。
图2为本发明的564G2D3抗CK6单克隆抗体和市售抗体在7个梯度浓度下效价检测的统计图。
图3为抗CK6重组兔单克隆抗体564G2D3作为一抗的免疫印记(Western blotting)检测结果验证其对CK6蛋白的识别能力。
具体实施方式
为进一步阐述本发明所采取的技术手段及其效果,以下结合实施例和附图对本发明作进一步地说明。可以理解的是,此处所描述的具体实施方式仅仅用于解释本发明,而非对本发明的限定。
实施例中未注明具体技术或条件者,按照本领域内的文献所描述的技术或条件,或者按照产品说明书进行。所用试剂或仪器未注明生产厂商者,均为可通过正规渠道商购获得的常规产品。人体组织样本是经过福尔马林固定、石蜡包埋的人体组织样本,均进行了病理证实,并有患者知情同意书。
实施例1
本实施例为抗CK6重组兔单克隆抗体的制备和筛选,步骤包括:
(1)抗原制备
CK6抗原具体序列如下所示的SEQ ID NO:1。
SEQ ID No. 1:YTTTSSSSRKSYKH。
上述多肽序列是通过对CK6分子序列进行分析,依据CK6蛋白分子在细胞膜上的结构、抗原性、组成氨基酸的亲疏水性以及二级结构后选择出来的。人工合成SEQ ID NO:1所示序列的多肽,并将合成的多肽作为免疫兔子用的抗原。免疫时,将SEQ ID NO:1所示序列的多肽
经与KLH或OVA偶联后制备成完全抗原,作为免疫原免疫兔子。
(2)免疫
将含有SEQ ID NO:1的多肽序列(CK6抗原)的完全抗原分别与完全弗氏佐剂(1:1)混合并乳化,采用皮下注射方法分别免疫多只新西兰大白兔,间隔两周后将含有上述序列(SEQ ID NO:1所示的多肽)的CK6抗原与不完全弗氏佐剂(1:1)乳化进行第二次和第三次免疫。三次免疫后取血以ELISA法梯度稀释测定血清效价;选取SEQ ID NO:1抗原免疫抗体效价最高的兔子进行下一步的细胞融合。
(3)细胞融合
提前准备鼠来源的sp2/0骨髓瘤细胞,使融合时该sp2/0骨髓瘤细胞
处于对数生长期。取已免疫兔脾脏,制成淋巴细胞单细胞悬液;兔脾淋巴细胞与所述骨髓瘤细胞混合,滴加50%PEG1500,加入IMDM培养基,离心弃上清后加入HAT培养基轻柔悬浮混匀,定容至800mL后分装于96孔板中,置于37℃、5%CO2恒温培养箱中进行培养。融合6-9天后观察96孔板中融合细胞状态,换液用HT继续置于37℃、5%CO2恒温培养箱中培养。
(4)筛选和克隆
融合7-10天后使用CK6抗原(SEQ ID NO:1)进行ELISA测试来筛选克隆细胞。标记好相应细胞株号,对阳性孔细胞进行有限稀释,直至ELISA测定96孔板全板结果为阳性。挑选出阳性值高的单克隆稳定株,得到分泌特异单克隆抗体的杂交瘤细胞株,记录为564G2D3。
(5)将筛选好的杂交瘤细胞株分泌的抗体进行测序
依据试剂TriZol说明书,从564G2D3杂交瘤细胞中分离出总RNA,依据TIANScript第一链cDNA合成试剂盒说明书,将总RNA逆转录成cDNA,利用特异性引物(重链可变区引物,VH-F :AGACTGGGCTGCGCTGGCTTC,VH-R:GTGAGGGTGCCCGAG;轻链可变区引物:VK-F :ATGGACAYGAGGGCCCCCACTC,VK-R: GGTGGGAAGATGAGGACAGTAGG)扩增获得抗体重链可变区和抗体轻链可变区的核苷酸序列,然后将抗体重链可变区和抗体轻链可变区的核苷酸序列克隆至真核表达载体(InvivoGen,pfuse-rchg,pfuse2-rclk1)中,准备进行细胞转染。
(6)细胞转染与筛选
提前准备好待转染用的293细胞,离心换新鲜的培养基后分别放入24孔板中,按所需要的数量每孔1.5ml,密度为3×106个/ml。
将所述真核表达载体与PEI按比例1:6混合后加入到准备好的293细胞中,置于37℃、5%CO2的摇床中培养。培养3-5天后将转染的细胞上清与对应抗原进行ELISA检测来筛选阳性孔,再将阳性孔的细胞上清继续进行免疫组织化学法检测,如果免疫组织化学法检测阳性则确认测出的抗体序列正确。
(7)细胞上清单抗的制备与纯化
将确认阳性的表达载体进行大量的细胞转染,继续培养3-5天后,收取细胞悬液,离心后取上清,利用亲和层析法进行纯化。纯化后的单抗浓度测定、分装、于4-8℃冰箱中保存。
最终,564G2D3抗CK6重组兔单克隆抗体的重链可变区氨基酸序列由SEQ IDNo.2所示的DNA序列所编码,抗CK6重组兔单克隆抗体的轻链可变区氨基酸序列由SEQ ID No.3所示的DNA序列所编码。
SEQ ID No.2-3具体序列如下:
SEQ ID No.2:
cagtcgctggaggagtccgggggtcgcctggtcacgcctgggacacccctgacactcacctgcacagcctctggattctccctcagtagatatacaatgggctgggtccgccaggctccagggaaggggctggaacacatcggattcattgatggtggtggtagcgcagcctacgcgagctgggcgaaaggccgattcaccatctccaaaacctcgtcgaccacggtggatctgaaaatgaccagtccgacaaccgaggacacggccacctatttctgtgccaagtttgatgataattataaaacctatcacatctggggcccaggcaccctggtcaccgtctcctcaa。
SEQ ID No.3:
gccgtcgtgatgacccagactgcatcgcccgtgtctgcagctgtgggaggcacagtcaccatcgattgccaggccagtcagagtattagtagtaactacttatcctggtatcagcagaaaccagggcagcgtcccaagctcctgatctacagggcatccactctggcatctggggtcccatcgcggttcaaaggcagtggatctgggacagagttcactctcaccatcagtggcgtgcagtgtgacgatgctgccacttactactgtctatacggtgattatattagtacgagtggtgatgttttcggcggagggaccgaggtggtggtcaaag。
将获得的碱基序列翻译成氨基酸序列,分析,获得564G2D3抗CK6重组兔单克隆抗体的重链可变区氨基酸序列为SEQ ID No.4所示,所述抗CK6重组兔单克隆抗体的轻链可变区氨基酸序列为SEQ ID No.5所示。
SEQ ID No.4-5具体序列如下:
SEQ ID No.4:
QSLEESGGRLVTPGTPLTLTCTASGFSLSRYTMGWVRQAPGKGLEHIGFIDGGGSAAYASWAKGRFTISKTSSTTVDLKMTSPTTEDTATYFCAKFDDNYKTYHIWGPGTLVTVSS。
SEQ ID No.5:
AVVMTQTASPVSAAVGGTVTIDCQASQSISSNYLSWYQQKPGQRPKLLIYRASTLASGVPSRFKGSGSGTEFTLTISGVQCDDAATYYCLYGDYISTSGDVFGGGTEVVVK。
实施例2
本实施例为抗CK6重组兔单克隆抗体作为一抗的免疫组化检测,方法如下:
(1)样本切片准备:将经福尔马林固定石蜡包埋后的人肺鳞癌切片于60℃恒温箱中烤片1-2h,保存备用。
(2)切片脱蜡:石蜡切片先置于新鲜二甲苯中进行脱蜡,浸泡2次,每次10 min。
(3)切片水化:依次经过无水乙醇,无水乙醇,95%乙醇,85%乙醇,70%乙醇浸泡5分钟进行水化,后纯化水冲洗2次,每次3 min。
(4)抗原修复:推荐使用高温热修复法修复3min(如果使用自动修复仪可设置98℃高温修复20 min),切片自然冷却至室温后用免疫组化笔将待测组织圈起来,纯化水冲洗2次,每次3 min。
(5)内源性过氧化物酶灭活:滴适量内源性过氧化物酶阻断剂完全覆盖组织,室温孵育10 min后,纯化水冲洗2次,每次3 min,PBST冲洗一次。
(6)一抗孵育:分别在两个样品中加入100μL的10ng/mL 564G2D3抗CK6重组兔单克隆抗体和市售CK6抗体完全覆盖组织,置于37℃恒温箱中孵育1h,PBST冲洗3次,每次5min。
(7)二抗孵育:依照所用二抗染色系统DAB染色液试剂盒的说明书进行二抗孵育,孵育完毕后PBST冲洗片3次,每次5min,纯化水冲洗1次。
(8)DAB显色:依照所用DAB染色液试剂盒说明书进行配制DAB显色液,滴适量配制好的DAB显色液至完全覆盖组织,待颜色无加深终止染色,纯化水冲洗3次。
(9)苏木素复染:依照苏木素厂家说明书操作步骤及建议对切片进行复染,PBST或自来水冲洗返蓝。
(10)脱水透明:依次浸泡70%,85%,95%,100%,100%梯度酒精,每次3 min;2次二甲苯透明,每次5 min。
(11)封片:用中性树胶对样品进行封片。
由图1结果所示,CK6蛋白在人肺鳞癌组织中呈特异性细胞质染色,且564G2D3克隆的CK6抗体染色效果比市售CK6抗体更好(染色颜色更深)。由此说明,本发明的564G2D3克隆的CK6重组兔单克隆抗体由于其特异性好,阳性信号强等特点,在IHC染色中染色信号更强,有利于准确检测区分癌症。
实施例3
本实施例为对564G2D3抗CK6重组兔单克隆抗体亲和力的测定,测定方法如下:
(1)从4℃中取出标记好的CK6的多肽(SEQ ID NO:1),恢复至室温。稀释至浓度1μg/ml,按100μL/孔加到96孔酶标板上4℃孵育过夜,随后用2% BSA进行4℃封闭过夜。
(2)将564G2D3克隆的CK6重组兔单克隆抗体稀释成初始浓度为0.5μg/mL,并依次进行2倍梯度稀释,共设7个浓度梯度进行对比。
(3)分别将稀释好的抗CK6重组兔单克隆抗体按照100μL/孔添加至有多肽的96孔酶标板上,盖上封板膜,37℃恒温孵育1h,使反应达到平衡。
(4)反应结束后取出酶标板,弃掉液体,纯化水冲洗5次,拍干水分。
(5)按照二抗使用说明书稀释HRP标记羊抗兔IgG,以100μL/孔加入酶标板中,37℃恒温孵育1h,使反应达到平衡。
(6)反应结束后取出酶标板,弃掉液体,纯化水冲洗5次,拍干水分。
(7)按100μL/孔加入TMB显色液,室温反应6分钟。
(8)反应结束后,按50μL/孔加入2M H2SO4终止显色。
(9)在酶标仪上于450nm读取OD值,整理数据,分析结果如下图2所示。
结果显示,在7个浓度梯度试验中,本发明的564G2D3克隆的抗CK6重组兔单克隆抗体对CK6蛋白分子的亲和力强、灵敏度高,可在较低抗体浓度(尤其在浓度介于31.25ng/mL-125 ng/mL)条件下达到较高OD值,如此可提高检测准确性的同时节约检测成本。
实施例4
本实施例为抗CK6重组兔单克隆抗体564G2D3作为一抗的免疫印记(Westernblotting)检测,方法如下:
(1)选择相应细胞(人皮肤组织和人扁桃体组织细胞)裂解液的PVDF膜进行活化,甲醇活化1min,用纯水洗膜2次后再用TBST洗涤3次;封闭:将膜置于5%BSA配置成的封闭液中,室温混摇2h;
(2)一抗孵育:将564G2D3抗体稀释成0.5μg/mL浓度,将封闭好的膜放入对应的已稀释抗体中,置4℃混摇孵育过夜;
(3)取出膜放入TBST液中洗涤3次(2×5min+1×10min);
(4)二抗孵育:将HRP-抗兔IgG用FG液以1:5000稀释,混匀后加入膜条,室温混摇1h;
(5)取出膜条放在TBST液中洗涤4次(3×5min+1×8min);
(6)底物:将用纯水5倍稀释的等量鲁米诺试剂和过氧化氢溶液,于同一容器内混匀,加入膜条,孵育2min;
(7)曝光:把底片放在暗盒中,根据荧光强度分别对X光胶片作不同时间段的曝光;然后按照1min显影,清洗,1min定影的顺序进行操作,最后清洗并晾干;结果如图3所示。
图3中泳道1:人皮肤组织裂解物H.skin,泳道2:人扁桃体组织裂解物H.tosil;条带大小:42 kDa。
由图3结果可见,在泳道中,564G2D3抗CK6重组兔单克隆抗体能够特异性的识别人皮肤组织裂解物H.skin和人扁桃体组织裂解物H.tosil中过量表达的CK6蛋白,分子量在60kDa左右,说明本发明的564G2D3克隆的CK6重组兔单克隆抗体能够高特异性的识别CK6蛋白。
最后应说明的是:以上各实施例仅用以说明本发明的技术方案,而非对其限制;尽管参照前述各实施例对本发明进行了详细的说明,本领域的普通技术人员应当理解:其依然可以对前述各实施例所记载的技术方案进行修改,或者对其中部分或者全部技术特征进行等同替换;而这些修改或者替换,并不使相应技术方案的本质脱离本发明各实施例技术方案的范围。
Claims (9)
1.一种抗CK6重组兔单克隆抗体,其特征在于,包括重链可变区和轻链可变区,所述重链可变区的氨基酸序列如SEQ ID No.4所示;所述轻链可变区的氨基酸序列如SEQ ID No.5所示。
2.编码基因,其特征在于,用于编码权利要求1所述的抗CK6重组兔单克隆抗体。
3.根据权利要求2所述的编码基因,其特征在于,其包括:如SEQ ID No.2所示的DNA序列,以用于编码所述抗CK6重组兔单克隆抗体的重链可变区;以及如SEQ ID No.3所示的DNA序列,以用于编码所述抗CK6重组兔单克隆抗体的轻链可变区。
4.一种核酸分子,其特征在于,其含有权利要求2或3所述的编码基因。
5.一种表达载体,其特征在于,其含有权利要求4所述的核酸分子。
6.一种抗CK6重组兔单克隆抗体的制备方法,其特征在于,其采用权利要求5所述的表达载体对细胞进行转染,转染后继续培养细胞,收集细胞上清液并纯化,得到所述抗CK6重组兔单克隆抗体。
7.权利要求1所述的抗CK6重组兔单克隆抗体、权利要求2或3所述编码基因、权利要求4所述的核酸分子或权利要求5所述的表达载体在制备CK6检测试剂盒中的应用。
8.一种CK6检测试剂盒,其特征在于,其包括权利要求1的抗CK6重组兔单克隆抗体和免疫组织化学检测试剂。
9.根据权利要求8所述的CK6检测试剂盒,其特征在于,其包括:抗CK6重组兔单克隆抗体、HRP酶标二抗、EDTA修复液、过氧化氢酶封闭液、DAB浓缩液、DAB缓冲液、苏木素和返蓝液。
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