CN116063537B - 一种高亲和力结合tet(x2)蛋白的单克隆抗体及其产品和应用 - Google Patents
一种高亲和力结合tet(x2)蛋白的单克隆抗体及其产品和应用 Download PDFInfo
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Abstract
本发明提供了一种高亲和力结合TET(X2)蛋白的单克隆抗体及其产品和应用,属于单克隆抗体技术领域。本发明首先人工合成核苷酸序列如SEQ ID NO.1所示,氨基酸序列如SEQ ID NO.2所示的截短型TET(X2)抗原,然后诱导、表达、纯化获得抗原蛋白,接着进行动物免疫,最终获得重链Fd段核苷酸序列如SEQ ID NO.9所示,氨基酸序列如SEQ ID NO.17所示,轻链核苷酸序列如SEQ ID NO.10所示,氨基酸序列如SEQ ID NO.18所示的单克隆抗体。本发明中的单抗与TET(X2)蛋白亲和力值高达6.2×10‑10nM,可有效应用于替加环素抗药性的快速检测中。
Description
技术领域
本发明属于单克隆抗体技术领域,尤其涉及一种高亲和力结合TET(X2)蛋白的单克隆抗体及其产品和应用。
背景技术
替加环素是第三代四环素家族的抗生素成员,常用于治疗耐碳青霉稀类多重耐药的革兰阴性菌,被认为是抗生素的“最后一道防线”。TET(X)家族是一类单加氧酶,目前已发现有14个成员,TET(X)家族成员之间的氨基酸序列相似性可高达80%。TET(X)家族不仅对四环素家族的全部抗生素具有较强的水解活性,而且是近年来发现了可以通过质粒转移的TET(X)家族成员基因,这可能会造成此基因在临床细菌建播撒及替加环素抗性的蔓延,导致临床细菌耐药性形势发生恶化。TET(X2)是TET(X)家族中最早发现的成员之一,最早发现于黄杆菌科细菌的固有染色体基因组中。目前学术界认为后来发现TET(X)家族的成员可能是由TET(X2)进化而来。因此在临床上快速检测临床菌株的TET(X2)对于临床抗生素用药及细菌耐药性的防控具有重要意义。
目前,临床微生物实验室细菌耐药性的常规检测方法多为肉汤微量稀释法、纸片法等,存在耗时长的缺点。因此,细菌耐药性快速检测新技术的研发已成为检验与临床关注的焦点。针对病原微生物耐药性的快速检测方法不断涌现,如:基质辅助激光解吸电离飞行时间质谱技术(MALDI-TOFMS),光谱技术,电化学生物传感器,综合性分子诊断平台,宏基因组测序等等。虽耗时短,但是这些方法存在费用高昂,且对仪器设备及操作者要求过高的问题,因此,尚不容易普及,也难以及时指导抗感染治疗。
相比之下,基于抗原-抗体反应的快速检测方法因具有敏感性高、特异性好,且检测成本相对较低、对设备和操作者的技术要求相对较低等优点,目前已经在临床上广泛应用,但是目前尚未有将该方法应用于TET(X2)蛋白检测中的相关报道。
发明内容
为了解决上述技术问题,本发明提供了一种高亲和力结合TET(X2)蛋白的单克隆抗体及其产品和应用。本发明首先人工合成核苷酸序列如SEQ ID NO.1所示,氨基酸序列如SEQ ID NO.2所示的截短型TET(X2)抗原,然后诱导、表达、纯化获得抗原蛋白,接着使用该抗原蛋白进行动物免疫,最终获得重链Fd段核苷酸序列如SEQ ID NO.9所示,氨基酸序列如SEQ ID NO.17所示,轻链核苷酸序列如SEQ ID NO.10所示,氨基酸序列如SEQ ID NO.18所示的单克隆抗体。本发明中的单抗与TET(X2)蛋白亲和力值高达6.2×10-10nM,可有效应用于替加环素抗药性的快速检测中。
为了实现上述目的,本发明采用了以下技术方案:
本发明提供了一种高亲和力结合TET(X2)蛋白的单克隆抗体,所述重链可变区包含CDR1、CDR2、CDR3,其中CDR1的核苷酸序列如SEQ ID NO.3所示,氨基酸序列如SEQ IDNO.11所示;CDR2的核苷酸序列如SEQ ID NO.4所示,氨基酸序列如SEQ ID NO.12所示;CDR3的核苷酸序列如SEQ ID NO.5所示,氨基酸序列如SEQ ID NO.13所示;所述轻链可变区包含CDR1、CDR2、CDR3,其中CDR1的核苷酸序列如SEQ ID NO.6所示,氨基酸序列如SEQ ID NO.14所示;CDR2的核苷酸序列如SEQ ID NO.7所示,氨基酸序列如SEQ ID NO.15所示;CDR3的核苷酸序列如SEQ ID NO.8所示,氨基酸序列如SEQ ID NO.16所示。
优选的,所述单克隆抗体重链Fd段的核苷酸序列如SEQ ID NO.9所示,氨基酸序列如SEQ ID NO.17所示;所述单克隆抗体轻链的核苷酸序列如SEQ ID NO.10所示,氨基酸序列如SEQ ID NO.18所示。
本发明还提供了一种扩增所述单克隆抗体的引物组合,其中所述重链Fd段上游引物的核苷酸序列如SEQ ID NO.19所示,下游引物的核苷酸序列如SEQ ID NO.20所示;所述轻链上游引物的核苷酸序列如SEQ ID NO.21所示,下游引物的核苷酸序列如SEQ ID NO.22所示。
本发明还提供了一种包含所述单克隆抗体或所述引物组合的试剂盒。
本发明还提供了一种与所述单克隆抗体特异性结合的截短型抗原,其核苷酸序列如SEQ ID NO.1所示,氨基酸序列如SEQ ID NO.2所示。
本发明还提供了一种所述单克隆抗体或所述引物组合或所述试剂盒或所述截短型抗原在TET(X2)蛋白检测中的应用。
本发明还提供了一种所述单克隆抗体或所述引物组合或所述试剂盒或所述截短型抗原在替加环素耐药性检测中的应用。
与现有技术相比,本发明具有如下技术效果:
本发明中基于抗原-抗体反应的快速检测方法不仅具有敏感性高、特异性好、检测成本低、对设备和操作者的技术要求低等优点,而且本发明中的单克隆抗体与TET(X2)蛋白亲和力值还高达6.2×10-10nM,亲和力十分高,可有效应用于替加环素抗药性的快速检测中。
附图说明
图1为本发明实施例1中纯化截短型TET(X2)抗原蛋白的SDS-PAGE图;
图2为本发明实施例2中单克隆抗体的WB特异性鉴定结果图,其中M表示蛋白质marker,其条带从上至下依次为:100kDa、75kDa、60kDa、45kDa、35kDa和25kDa;
图3为本发明实施例3中抗体基因的扩增结果,其中M为DNA ladder,H和L分别指轻链和重链PCR扩增的结果;
图4为本发明实施例4中抗体亲和力SPR检测图。
具体实施方式
以下实施例用于说明本发明,但不用来限制本发明的范围。在不背离本发明精神和本质的情况下,对本发明方法、步骤或条件所作的修改或替换,均属于本发明的范围。以下实施例中使用的试剂、试剂盒和仪器均可由市售获得,实施例中使用的方法如无特别说明,与常规使用的方法一致。
下面结合实施例,对本发明的技术方案进行进一步详细阐述。
实施例1TET(X2)抗原蛋白的表达及纯化
(1)TET(X2)抗原蛋白表达设计方案:
TET(X)家族蛋白长度约380-390AAs,家族成员之间的氨基酸序列相似性在80%以上。根据TET(X2)的序列,采用大肠杆菌系统表达蛋白的110-362AA截短型TET(X2)。直接合成基因,N端添加GSlinker及6*His标签,通过NdeI/EcoRI酶切位点克隆入pET28b载体。
(2)合成的截短型TET(X2)基因序列:
ATGGGCAGCAGCCATCATCATCATCATCATCACAGCAGCGGCCTGGTGCCGCGCGGCAGCCATATGTTCGATAACCCGGAAATTAATCGCAATGATCTGCGCGCCATTCTGCTGAATAGCCTGGAAAATGATACCGTTATTTGGGATCGTAAACTGGTTATGCTGGAACCGGGTAAAAAGAAATGGACCCTGACCTTTGAAAATAAGCCGAGTGAAACCGCCGATCTGGTTATTCTGGCAAATGGCGGTATGAGCAAAGTTCGTAAATTTGTGACCGATACCGAAGTTGAAGAAACCGGTACCTTTAATATTCAGGCCGATATTCATCAGCCGGAAATTAACTGCCCGGGCTTTTTCCAGCTGTGCAATGGCAATCGCCTGATGGCCAGCCATCAGGGCAATCTGCTGTTTGCAAATCCGAATAATAATGGTGCCCTGCATTTTGGTATTAGCTTTAAAACCCCGGATGAATGGAAAAATCAGACCCAGGTTGATTTTCAGAATCGTAATAGCGTGGTGGATTTTCTGCTGAAAGAATTTTCAGATTGGGATGAACGCTATAAAGAACTGATTCATACCACCCTGAGCTTTGTGGGTCTGGCAACCCGCATTTTTCCGCTGGAAAAACCGTGGAAAAGTAAACGCCCGCTGCCGATTACCATGATTGGCGATGCCGCCCATCTGATGCCGCCGTTTGCCGGCCAGGGCGTGAATAGTGGCCTGGTTGATGCACTGATTCTGAGTGATAATCTGGCAGATGGTAAATTCAATAGCATTGAAGAAGCCGTGAAAAATTATGAACAGCAGATGTTTATCTACGGCAAAGAAGCA(SEQ ID NO.1)。
(3)表达的截短型TET(X2)蛋白氨基酸序列
MGSSHHHHHHHSSGLVPRGSHMFDNPEINRNDLRAILLNSLENDTVIWDRKLVMLEPGKKKWTLTFENKPSETADLVILANGGMSKVRKFVTDTEVEETGTFNIQADIHQPEINCPGFFQLCNGNRLMASHQGNLLFANPNNNGALHFGISFKTPDEWKNQTQVDFQNRNSVVDFLLKEFSDWDERYKELIHTTLSFVGLATRIFPLEKPWKSKRPLPITMIGDAAHLMPPFAGQGVNSGLVDALILSDNLADGKFNSIEEAVKNYEQQMFIYGKEA(SEQ ID NO.2)。
Features:
加粗字体部分[1-13AA]:GS linker with 6*His tag
下划线部分[14-19AA]:Thrombin site
(4)截短型TET(X2)蛋白表达及纯化
带有pTET(X2)质粒的大肠埃希菌DH5α于37℃220rpm震荡培养16小时,12000g离心5min,细菌沉淀采用SteadyPure质粒提取试剂盒(湖南艾瑞科生物)提取pTET(X2)质粒,具体操作参照说明书进行。
感受态大肠杆菌BL21(DE3)从-80℃取出后,放置冰上30min,加入pTET(X2)质粒42℃热激45s,加入900μL 37℃预热的SOC培养基,37℃150rpm震荡1h,均匀涂布于含50μg/L卡那霉素的平板中,过夜培养,挑选阳性克隆,通过PCR确定截短型TET(X2)蛋白表达菌株构建成功。
挑取划线平板上单克隆接种于含有50μg/L卡那霉素的LB培养液中;37℃220rpm振摇至菌体OD600为0.6-0.8;向培养物中加入IPTG至终浓度分别为1mmol/L,诱导融合蛋白表达,继续培养6h;菌液经4℃6,000g离心30min,细菌沉淀用缓冲液A(50mmol/L Tris,300mmol/L NaCl,pH 7.4)重悬离心后沉淀,冰水浴中超声波破碎10min(功率750W,工作1秒,间歇2秒)。4℃6,000g离心30min后,上清液过Ni柱,用洗涤缓冲液(50mmol/L Tris,300mmol/L NaCl,50mmol/L Imidazole,pH7.4)洗涤Ni-柱三次后,用洗脱缓冲液(50mmol/LTris,300mmol/L NaCl,500mmol/L Imidazole,pH7.4)洗脱蛋白。取2μg纯化蛋白进行SDS-PAGE。结果如图1所示。
实施例2小鼠单抗的制备
(1)动物免疫及间接ELISA的建立
以实施例1中的截短型TET(X2)蛋白作为免疫原,免疫4只6周龄BALBc小鼠,免疫剂量为100μg只,免疫方法为背部皮下多点注射,每2周免疫1次,首免为免疫原加等体积弗氏完全佐剂,二免和三免为免疫原加等体积弗氏不完全佐剂。三免7天后,小鼠眼球采血,收集血清。融合前3天,腹腔注射纯抗原进行加强免疫。具体过程如表1所述。间接ELISA最佳抗原、抗体工作浓度及阴阳临界值的确定采用方阵滴定法确定。
表1小鼠免疫过程
免疫时间 | 剂量 |
一免(Day1) | 0.05mg只 |
二免(Day14) | 0.025mg只 |
三免(Day28) | 0.025mg只 |
抗血清效价检测(Day35) | ELISA检测血清效价大于1:32000 |
四免(Day42) | 0.025mg只 |
抗血清效价检测(Day49) | ELISA检测血清效价大于1:32000 |
(2)细胞融合及亚克隆筛选
将4份免疫小鼠血清分别进行1:4000、1:8000、1:16000稀释,按照已建立的间接ELISA测定血清效价,效价>104以上即可进行融合。加强免疫3天后取小鼠脾细胞与SP20骨髓瘤细胞进行融合,融合后取上清用已建立的ELISA检测,然后采用有限稀释法及方阵滴定法对阳性杂交瘤细胞进行亚克隆,采用已建立的ELISA进行检测是否亚克隆成功。将阳性孔细胞按照上述方法连续克隆3次,选择OD值高、细胞活力好且能稳定分泌抗体的阳性杂交瘤孔,进行扩大培养并及时冻存。
(3)腹水制备及单克隆抗体效价
给7周龄BALBc小鼠腹腔注射0.5mL无菌液体石蜡,7-10天后每只小鼠腹腔注射1mL杂交瘤细胞(1×106个/mL),14天后采集腹水,离心取上清,采用ProteinLResin试剂盒进行纯化,并用BCAProteinAssayKit试剂盒测定纯化腹水浓度。按照间接ELISA对单克隆抗体进行效价测定,以纯化的截短型TET(X2)蛋白作为阳性对照,SP20细胞上清和阴性血清作为阴性对照,每个样本重复3次。
(4)单克隆抗体特异性的Western blot(WB)鉴定
取5μg纯化的截短型TET(X2)蛋白、蛋白表达宿主菌BL21 DE3、鲍曼不动杆菌、肺炎克雷伯菌、铜绿假单胞菌和金黄色葡萄球菌的1mL过夜培养物的菌体总蛋白,上样进行SDS-PAGE,转膜后用含5%牛血清白蛋白的TBST缓冲液[Tris-HCl(1mol/L,pH7.5):50mL,NaCl:8g,KCl:0.2g,吐温80 0.5mL,加蒸馏水定容至1L]室温封闭1h,以杂交瘤细胞上清(1:2000)为一抗,于低温摇床上4℃过夜孵育。次日,洗膜后,加入辣根过氧化物酶标记Goat Anti-Mouse IgG(1:5000)为二抗,用eECL高灵敏度发光试剂进行检测。
检测结果如图2所示,其中M为蛋白质marker,分子量从上至下依次为:250、150、100、70、50、40、35、20kDa;泳道1-6分别为截短型TET(X2)蛋白、蛋白表达宿主菌BL21 DE3、鲍曼不动杆菌、肺炎克雷伯菌、铜绿假单胞菌和金黄色葡萄球菌的1mL过夜培养物的菌体总蛋白。由图2可以看出,在40-50kDa位置,完整的TET(X)家族蛋白均出现条带,表明所获得的单克隆抗体仅与TET(X2)蛋白结合,而不与其他菌株的蛋白结合,表明所获得的抗体具有较高的特异性。
实施例3抗体基因序列的测定
(1)PCR引物
重链Fd5’引物
上游引物H5:5’-GAAGTGMAGCTCGAGGAGTCTGGGGGA-3’(SEQ ID NO.19);
下游引物H3:5’-AGGCTTACTAGTACAAATCCCTGGGCACAAT-3’(SEQ ID NO.20);
轻链引物:
上游引物L5:5’-CCTCTAGACAGGCTGTTGTGACTCAGGAATC-3’(SEQ ID NO.21);
下游引物L3:5’-GCGCCGTCTAGAATTAACACTCATTCCTGTTGAA-3’(SEQ ID NO.22)。
抗体基因的扩增结果如图3所示,扩增出与目的基因大小一致的PCR产物。
(2)抗体基因测序结果
重链CDR1:ACCTTTTCTGATAAG(SEQ ID NO.3);
重链CDR2:TACTCTCTGGATAACTTTTACTCTCCTAGGTATGATAACGACAAGGGGCTG(SEQ IDNO.4);
重链CDR3:TTTGAACGAGATGAGTCTACC(SEQ ID NO.5);
重链Fd段的碱基序列(共726bp)
ATGAACTTTGTGCTCACCTTGATTGCCCTTGCCCTCATTTTAAAATACGTCCAGTGTGAAGTACAACTGGTGGAGTCTGGGGGAGACTACGTGAAGCCTGGAGGGTCCCTGAAACTCTCCTGTGCAGCCTCCGGATTCACTTTCAGTACCTTTTCTGATAAGTGGGTTCGGCAGACTCCGGAGAGGAAATTGGAGTGGGTCGCAGCGTACTCTCTGGATAACTTTTACTCTCCTAGGTATGATAACGACAAGGGGCTGCGATTCACCGTCTCCAGAGACAATGCCAAGAACACCCTATACCTGCAGATGAGCAGTCTGAGGTCTGAGGACACGGCCATATATTACTGTGTGAGATTTGAACGAGATGAGTCTACCTGGGGCCAAGGGACTCTGGTCACTGTCTCTGCTGCCAAAACGACACCCCCATCTGTCTATTCTCTGGCCCCTGGATCTGCTGCCCAAACTAACTCCATGGTGACCCTGGGATGCCTGGTCAAGGGCTATTTCAAGGAGCCAGTGACAGTGACCTGGAACTCTGGATCCCTGTCCAGCGGTGTGCACACCTTCCCAGCTGTCCTGCAGTCTGACATTTACACTTTGAGCAGCTCAGTGACTGTCCCCTCCAGCACCTGGCCC AGGGAGACCGTCACCTGCAACGTTGCCCACCCGCTGAGCAGCACCAAGGTGGACAAGAAAATTGTGCCCAGGGATTGTACTAGTAAGCCT(SEQ ID NO.9)。
轻链CDR1:CCTCGCTCTAAACTCAACCACCTCGGGTTCATTTCTACTCTTTAT(SEQ ID NO.6);
轻链CDR2:CTTAAAATGACTGAATCT(SEQ ID NO.7);
轻链CDR3:GAACTCGATCTTTATCTTCAAGAGACG(SEQ ID NO.8);
轻链的碱基序列(共660bp):
ATGGATATTGTGCTGACGCAGGCTGCCGATTCCAATCCAGTCACTCTTGGAACATCAGCTTCCATCTCCTGCGACCCTCGCTCTAAACTCAACCACCTCGGGTTCATTTCTACTCTTTATTGGTATCTGCAGAGGCCAGGCCAGTCTCCTCAGCTCCTGATTTCTTTTCTTAAAATGACTGAATCTGGAGTCCCAGACAGGTTCAGTAGCAGTGGGTCAGGAACTGATTTCACACTGAGAATCAGCAGAGTGGAGGCTGAGGATGTGGGTGTTTATTACTGTGAACTCGATCTTTATCTTCAAGAGACGTTCGGAGGGGGGACCAAGCTGGAAATAAAACGGGCTGATGCTACTCCAACTGTATCCATCTTCCCACCATCCAGTGAGCAGTTAACATCTGCTGGTGCCTCAGTCGTGTGCTTCTTGAACAACTTCTACCCCAGTGACATCAATGTCAAGTGGAAGATTGATGGCAGTGAACGACAAAATGGCGTCATTAACAGTTGGACTGATCAGGACAGCAAAGACAGCACCTACAGCATGAGCAGCACCCTCACGTTGACCAAGGACCACTATGAACGACATAACAGCTATACCTGTGAGGCCACTCACGAGACATCAACTTCACCCATTGTCAAGAGCTTCAACAGGAATGAGAAT(SEQ ID NO.10)。
(3)抗体氨基酸序列
重链CDR1:TFSDK(SEQ ID NO.11);
重链CDR2:YSLDNFYSPRYDNDKGL(SEQ ID NO.12);
重链CDR3:FERDEST(SEQ ID NO.13);
重链Fd段的氨基酸序列:
MNFVLTLIALALILKYVQCEVQLVESGGDYVKPGGSLKLSCAASGFTFSTFSDKWVRQTPERKLEWVAAYSLDNFYSPRYDNDKGLRFTVSRDNAKNTLYLQMSSLRSEDTAIYYCVRFERDESTWGQGTLVTVSAAKTTPPSVYSLAPGSAAQTNSMVTLGCLVKGYFKEPVTVTWNSGSLSSGVHTFPAVLQSDIYTLSSSVTVPSSTWPRETVTCNVAHPLSSTKVDKKIVPRDCTSKP(SEQ ID NO.17);
轻链CDR1:DPRSKLNHLGFISTLY(SEQ ID NO.14);
轻链CDR2:FLKMTES(SEQ ID NO.15);
轻链CDR3:ELDLYLQET(SEQ ID NO.16);
轻链的氨基酸序列:
MDIVLTQAADSNPVTLGTSASISCDPRSKLNHLGFISTLYWYLQRPGQSPQLLISFLKMTESGVPDRFSSSGSGTD FTLRISRVEAEDVGVYYCELDLYLQETFGGGTKLEIKRADATPTVSIFPPSSEQLTSAGASVVCFLNNFYPSDINVKWKI DGSERQNGVINSWTDQDSKDSTYSMSSTLTLTKDHYERHNSYTCEATHETSTSPIVKSFNRNEN(SEQ ID NO.18)。
实施例4单抗的亲和力鉴定
采用抗鼠抗体捕获法在Fortebio(Octet RED 96蛋白质相互作用工作站,BLITZpro1.1.0.28)BLITZpro1.1.0.28)亲和力测定仪上检测抗体亲和力。测定时将抗鼠抗体Fc段的捕获抗体(AMC)生物探针浸泡于PBS中10min;将4μl单克隆抗体样品(15μg/mL)加载到AMC生物探针上,然后在PBS缓冲液中平衡100s,进一步将AMC探针与100nM TET(X2)蛋白进行结合反应,反应时间600s,之后将AMC探针转移至PBS中,进行解离反应,时间为1000s。实验完毕,扣除空白对照响应值,用软件进行1:1Langmuir结合模式拟合,计算抗原抗体结合的动力学常数。TET(X2)单克隆抗体与TET(X2)蛋白的反应曲线如图4所示。
拟合曲线并计算亲和力,得到抗体的亲和力值Kd为6.2×10-10nM,说明本发明中的单克隆抗体与TET(X2)蛋白的亲和力十分高,可有效应用于替加环素抗药性的快速检测中。
以上所述的实施例仅是对本发明的优选方式进行描述,并非对本发明的范围进行限定,在不脱离本发明设计精神的前提下,本领域普通技术人员对本发明的技术方案做出的各种变形和改进,均应落入本发明权利要求书确定的保护范围内。
Claims (5)
1.一种高亲和力结合TET(X2)蛋白的单克隆抗体,其特征在于,所述单克隆抗体包含重链可变区和轻链可变区;
所述重链可变区包含CDR1、CDR2、CDR3,其中CDR1的核苷酸序列如SEQ ID NO.3所示,氨基酸序列如SEQ ID NO.11所示;CDR2的核苷酸序列如SEQ ID NO.4所示,氨基酸序列如SEQID NO.12所示;CDR3的核苷酸序列如SEQ ID NO.5所示,氨基酸序列如SEQ ID NO.13所示;
所述轻链可变区包含CDR1、CDR2、CDR3,其中CDR1的核苷酸序列如SEQ ID NO.6所示,氨基酸序列如SEQ ID NO.14所示;CDR2的核苷酸序列如SEQ ID NO.7所示,氨基酸序列如SEQID NO.15所示;CDR3的核苷酸序列如SEQ ID NO.8所示,氨基酸序列如SEQ ID NO.16所示。
2.根据权利要求1所述的单克隆抗体,其特征在于,所述重链Fd段的核苷酸序列如SEQID NO.9所示,氨基酸序列如SEQ ID NO.17所示;所述轻链的核苷酸序列如SEQ ID NO.10所示,氨基酸序列如SEQ ID NO.18所示。
3.一种扩增权利要求2所述的单克隆抗体的引物组合,其特征在于,所述重链Fd段上游引物的核苷酸序列如SEQ ID NO.19所示,下游引物的核苷酸序列如SEQ ID NO.20所示;所述轻链上游引物的核苷酸序列如SEQ ID NO.21所示,下游引物的核苷酸序列如SEQ IDNO.22所示。
4.一种包含权利要求1~2任一所述的单克隆抗体或权利要求3所述的引物组合的试剂盒。
5.一种与权利要求1~2任一所述的单克隆抗体特异性结合的截短型抗原,其特征在于,其核苷酸序列如SEQ ID NO.1所示,氨基酸序列如SEQ ID NO.2所示。
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