CN116444670A - 一种抗人cd16鼠单抗及兔单抗蛋白序列测定及稳转细胞株建立 - Google Patents
一种抗人cd16鼠单抗及兔单抗蛋白序列测定及稳转细胞株建立 Download PDFInfo
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Abstract
本发明提供本发明提供一种抗CD16抗体,其重链CDR区氨基酸序列选自SEQ ID NO.1‑3,轻链CDR区氨基酸序列选自SEQ ID NO.4‑6。本发明属于抗体领域,本发明提供了一种抗人CD16鼠单抗的蛋白序列,并根据测序的蛋白序列设计了基因表达碱基序列,建立该基因的稳转CHO细胞株进行重组表达,表达纯化的抗体能够特异性识别人CD16抗原,抗体表达产量远高于传统的杂交瘤细胞制备抗体的产量。此外,将该抗体的小鼠IgG1置换成兔IgG,保留可变区不变,建立兔单抗的稳转CHO细胞株进行重组表达,表达纯化的抗体能够保持特异性识别人CD16抗原。
Description
技术领域
本发明属于抗体领域,具体涉及一种抗人CD16鼠单抗及兔单抗蛋白序列测定及稳转细胞株建立。
背景技术
CD16即FcγRIII,是一个分子量为50000-70000道尔顿的糖蛋白,属于Ig超家族成员。IgG的FcR家族(FcγR)由6个受体组成:FcγRI(CD64)、FcγRIIa(CD32a)、FcγRIIb(CD32b)、FcγRIIc(CD32c)、FcγRIIIa(CD16a)和FcγRIIIb(CD16b),其中CD16a主要负责触发NK细胞介导的ADCC效应。CD16分子广泛表达于NK细胞、嗜中性多形核白细胞、单个核细胞和巨噬细胞中,但在嗜碱细胞中不表达。抗CD16单克隆抗体可激活NK细胞抗体依赖性细胞介导的细胞毒性作用。
CD16触发的ADCC效应和分别由NK细胞、单核/巨噬细胞介导的吞噬作用是单克隆抗体发挥杀伤肿瘤细胞作用并达到治疗效果的主要免疫依赖机制[1]。研究人员拟通过激活或抑制NK细胞CD16受体调控单克隆抗体抗肿瘤反应,该研究成果有望应用于细胞免疫治疗的精准控制。
研究人员发现,流感疫苗肌肉内接种后,体内抗体-抗原免疫复合物的交联,下调了NK细胞CD16的表达,限制了NK的细胞脱颗粒作用,抑制了NK细胞对外源性细胞因子的反应能力[2]。在这一过程中,研究人员还证实了ADAM17类蛋白酶能够抑制CD16脱落,从而增强NK细胞细胞的细胞毒性功能。这项研究为CD16在NK细胞免疫治疗方面开辟了潜在的应用。
CD16分子作为一个经典靶点,是许多药物开发的候选位点,临床试验主要涉及肾移植及终末期肾病治疗、新冠病毒感染治疗、实体瘤治疗、心血管疾病治疗、呼吸道感染治疗等方向。
提高NK细胞对实体瘤的靶向特异性,为肿瘤患者提供更为有效的辅助治疗策略,是NK细胞药物开发的关键点。对CD16的作用机理、协同作用分子以及与信号通路等的研究,为NK细胞免疫疗法的开发提供了新方向。
利用杂交瘤的方式制备抗体时,由于杂交瘤不稳定,抗体基因容易丢失,此外,杂交瘤制备技术不会对目标抗体的基因序列及蛋白序列进行鉴定,不利于对目标抗体后续的生产,制备及研发工作。基因工程方法可以很好地解决杂交瘤方法中的问题,并且通过基因工程技术的改造,可以降低甚至消除人体对抗体的排斥反应,基因工程抗体的分子量较小,可以部分降低抗体的鼠源性,更有利于穿透血管壁,进入病灶的核心部位。通过基因工程方法进行抗体的制备必需的条件是明确抗体的序列,因此,挖掘更适用于CD16检测的单克隆抗体是当前更为合适的研发方向。
[1]Capuano C,Pighi C,Battella S,et al.Harnessing CD16-Mediated NKCell Functions to Enhance Therapeutic Efficacy of Tumor-TargetingmAbs.Cancers(Basel).2021May 20;13(10):2500.doi:10.3390/cancers13102500.
[2]Goodier MR,Lusa C,Sherratt S,et al.Sustained Immune Complex-Mediated Reduction in CD16 Expression after Vaccination Regulates NK CellFunction.Front Immunol.2016Sep 26;7:384.doi:10.3389/fimmu.2016.00384.
发明内容
为了解决上述问题,本发明首次公布了一种抗人CD16鼠单抗的蛋白序列,并通过设计基因与蛋白序列,建立该基因的稳转CHO细胞株进行重组表达,经验证,表达纯化的抗体能够特异性识别人CD16抗原,抗体表达产量远高于传统的杂交瘤细胞制备抗体的产量。此外,将该抗体的小鼠IgG1置换成兔IgG,保留可变区不变,建立兔单抗的稳转CHO细胞株进行重组表达,经验证,表达纯化的抗体能够保持特异性识别人CD16抗原。
术语:
本发明中,CDR(complementarity-determining regions,CDR)叫做互补决定区或互补决定簇。它位于免疫球蛋白的超变区,超变区是抗体的抗原结合位,与抗原决定簇的结构互补。一般包括CDR1、CDR2、CDR3。
本发明中,可变区指免疫球蛋白轻链和重链靠近N端氨基酸序列变化较大的区域称为可变区。
本发明中,重链指抗体中2条较长、相对分子量较大的相同的重链(H链);轻链指抗体中2条较短、相对分子量较小的相同的轻链(L链)。
本发明中,相似性指同源蛋白质的氨基酸序列中一致性和可取代氨基酸所占的比例。
本发明中,单克隆抗体指由单一B细胞克隆产生的高度均一、仅针对某一特定抗原表位的抗体。
本发明中,鼠源化抗体指将来源于免疫接种过的小鼠的B细胞与骨髓瘤细胞融合,得到的鼠杂交融合细胞分泌的抗体。
一方面,本发明提供了一种抗CD16抗体。
所述的抗CD16抗体包括重链和轻链,所述的重链包括重链链可变区,所述的轻链包括轻链可变区;所述的重链可变区包括HCDR1、HCDR2、HCDR3,所述的轻链可变区包括LCDR1、LCDR2、LCDR3,其中:
(1)HCDR1为SEQ ID NO.1所示的氨基酸序列,;
(2)HCDR2为SEQ ID NO.2所示的氨基酸序列;
(3)HCDR3为SEQ ID NO.3所示的氨基酸序列;
(4)LCDR1为SEQ ID NO.4所示的氨基酸序列;
(5)LCDR2为SEQ ID NO.5所示的氨基酸序列;
(6)LCDR3为SEQ ID NO.6所示的氨基酸序列。
具体地,所述的抗CD16抗体重链可变区氨基酸序列为SEQ ID NO.7,或与SEQ IDNO.7具有80%以上序列相似性的序列;所述的抗CD16抗体轻链可变区氨基酸序列选自SEQID NO.8,或与SEQ ID NO.8具有80%以上序列相似性的序列。
优选地,所述的抗CD16抗体重链可变区氨基酸序列为SEQ ID NO.7,轻链可变区氨基酸序列为SEQ ID NO.8。
具体地,所述的抗CD16抗体重链氨基酸序列为SEQ ID NO.9,或与SEQ ID NO.9具有65%以上序列相似性的序列;所述的抗CD16抗体重链氨基酸序列为SEQ ID NO.11,或与SEQ ID NO.11具有65%以上序列相似性的序列。
所述的抗CD16抗体轻链氨基酸序列为SEQ ID NO.10,或与SEQ ID NO.10具有65%以上序列相似性的序列,所述的抗CD16抗体轻链氨基酸序列为SEQ ID NO.12,或与SEQ IDNO.12具有65%以上序列相似性的序列。
优选地,所述的抗CD16抗体重链氨基酸序列为SEQ ID NO.9或SEQ ID NO.11,轻链氨基酸序列为SEQ ID NO.10或SEQ ID NO.12。
具体地,所述的抗CD16抗体为单克隆抗体。
优选地,所述的抗CD16抗体为鼠源化抗体。
另一方面,本发明提供了表达前述的抗CD16抗体的核苷酸序列。
所述的核苷酸序列可以是通过密码子简并性优化后的序列,用于编码前述的抗CD16抗体均可。
优选地,重链SEQ ID NO.9和SEQ ID NO.11对应的核苷酸序列分别为SEQ IDNO.14和SEQ ID NO.15所示、轻链SEQ ID NO.10和SEQ ID NO.12对应的核苷酸序列分别为SEQ ID NO.16和SEQ ID NO.17所示。
再一方面,本发明提供了包括前述的核苷酸序列的表达载体。
所述的表达载体可以是质粒、噬菌体、病毒。
又一方面,本发明提供了表达前述的抗CD16抗体或核苷酸序列或表达载体的细胞。
所述的细胞的作用在于可以高效表达抗CD16抗体。
具体地,所述的细胞可以是HEK293或CHO。
又一方面,本发明提供了制备前述的抗CD16抗体的方法,所述的方法中包括培养前述的细胞。
所述的方法中还可能包括抗体纯化步骤。
所述的抗体纯化步骤可以是沉淀法、广谱亲和纯化、抗原特异性纯化、离子交换法。
又一方面,本发明提供了对前述的抗CD16抗体进行荧光标记的方法,所述的方法中包括使用大分子染料或小分子染料对抗CD16抗体进行标记。
优选地,所述的大分子染料为APC,所述的小分子染料为FITC。
又一方面,本发明提供了前述的抗CD16抗体或核苷酸序列或表达载体或细胞在CD16检测中的应用,所述的应用为非疾病诊断或治疗的应用。
所述的应用为通过抗体抗原结合实现。
所述的应用可以是检测NK淋巴细胞中的CD16。
又一方面,本发明提供了前述的抗CD16抗体或核苷酸序列或表达载体或细胞在制备CD16检测试剂盒中的应用。
具体地,所述的试剂盒中还包括其他用于CD16检测的试剂,如:缓冲液、样品处理剂。
具体地,所述的试剂盒中还可以包括用于荧光标记抗体的分子。
进一步具体地,所述的分子可以是大分子染料或小分子染料。
优选地,所述的大分子染料为APC,所述的小分子染料为FITC。
又一方面,本发明提供了一种CD16检测方法,所述的检测方法通过前述的抗CD16抗体进行检测。
具体地,所述的检测方法中还可以包括样品前处理步骤。
所述的前处理步骤为样本的采集及处理。
优选地,所述的检测方法非疾病诊断或治疗方法。
又一方面,本发明提供了一种CD16检测试剂盒,所述的试剂盒中包括前述的抗CD16抗体或核苷酸序列或表达载体或细胞。
具体地,所述的试剂盒中还包括其他用于CD16检测的试剂,如:缓冲液、样品处理剂。
具体地,所述的试剂盒中还可以包括用于荧光标记抗体的分子。
进一步具体地,所述的分子可以是大分子染料或小分子染料。
优选地,所述的大分子染料为APC,所述的小分子染料为FITC。
本发明所取得的的技术效果:本发明公开了一种抗人CD16鼠单抗的蛋白序列,并根据测序的蛋白序列设计了基因表达碱基序列,建立该基因的稳转CHO细胞株进行重组表达,经验证,表达纯化的抗体能够特异性识别人CD16抗原,抗体表达产量远高于传统的杂交瘤细胞制备抗体的产量。此外,将该抗体的小鼠IgG1置换成兔IgG,保留可变区不变,建立兔单抗的稳转CHO细胞株进行重组表达,经验证,表达纯化的抗体能够保持特异性识别人CD16抗原。
附图说明
图1为抗人CD16抗体SDS凝胶电泳图。
图2为外购Biolegend鼠抗人CD16[B73.1]-FITC流式检测图。
图3为自制鼠抗人CD16[ZXCloneHCD16-04]-FITC流式检测图。
图4为自制兔抗人CD16[ZXCloneHCD16-05]-FITC流式检测图。
图5为外购Biolegend鼠抗人CD16[B73.1]-APC流式检测图。
图6为自制鼠抗人CD16[ZXCloneHCD16-04]-APC流式检测图。
图7为自制兔抗人CD16[ZXCloneHCD16-05]-APC流式检测图。
图8为外购Biolegend鼠抗人CD16[B73.1]效价检测图。
图9为自制鼠抗人CD16[ZXCloneHCD16-04]效价检测图。
图10为自制兔抗人CD16[ZXCloneHCD16-05]效价检测图。
图11为外购Biolegend鼠抗人CD16[B73.1]抗体特异性图。
图12为自制鼠抗人CD16[ZXCloneHCD16-04]抗体特异性图。
图13为自制兔抗人CD16[ZXCloneHCD16-05]抗体特异性图。
图14为对照CD16[B73.1]抗体特异性图。
具体实施方式
下面结合具体实施例,对本发明作进一步详细的阐述,下述实施例不用于限制本发明,仅用于说明本发明。以下实施例中所使用的实验方法如无特殊说明,实施例中未注明具体条件的实验方法,通常按照常规条件,下述实施例中所使用的材料、试剂等,如无特殊说明,均可从商业途径得到。
实施例1抗体的制备及检测
制备了两种抗体,自制鼠抗人CD16[ZXCloneHCD16-04],自制兔抗人CD16[ZXCloneHCD16-05]。
自制鼠抗人CD16[ZXCloneHCD16-04]重链氨基酸序列为SEQ ID NO.9,轻链氨基酸序列为SEQ ID NO.10;自制兔抗人CD16[ZXCloneHCD16-05]重链氨基酸序列为SEQ IDNO.11,轻链氨基酸序列为SEQ ID NO.12,常规方法设计表达相应抗体的核苷酸序列,重链SEQ ID NO.9和SEQ ID NO.11对应的核苷酸序列分别为SEQ ID NO.14和SEQ ID NO.15所示、轻链SEQ ID NO.10和SEQ ID NO.12对应的核苷酸序列分别为SEQ ID NO.16和SEQ IDNO.17所示。将核苷酸序列克隆至CHO稳转株后进行抗体稳转表达,进一步将构建载体导入CHO细胞。
转染和筛选:
(1)CHO细胞使用CHO CD培养基培养,2×105cells/mL接种,加4mM谷氨酰胺。细胞密度达到4×106cells/mL,活力大于95%,离心收集细胞,使用电转缓冲液重悬细胞,调整密度到1×107cells/mL。
(2)取无菌的EP管,加入0.8mL细胞悬液,加入5μg质粒,轻轻混匀,孵育15min,冰浴5min。电转仪设置参数为电压280V,脉冲次数3次,脉冲时间5ms,脉冲间隙0.784s,电转杯内径4mm。在每个电转杯中加入0.5mL细胞和质粒的混合液,放入电转仪启动电击程序,完成后迅速将电转杯中的细胞吸出,转移到装有20mL CHO CD培养基的125mL锥形瓶中,重复上述步骤电击第二个电转杯。轻轻混合摇瓶中的细胞,然后盖紧盖子置于37℃摇床中培养。
(3)48h后,将细胞转移到离心管中1000rpm离心5min。去掉上清,用20mL的含有2、5、20μM MSX的CHO CD培养基重悬细胞,计算细胞密度和活性。细胞密度在1.2×106cells/mL左右。
(4)将细胞转移到新的摇瓶中。从第七天开始,每两天观测细胞密度和活性。细胞密度从开始的1.2×106cells/mL往下降低,在第5-7天到达密度最低值,在第10-15天开始,细胞密度升高。当细胞密度升高到1×106cells/mL后,使用含有50μM MSX的CD OPM培养基将细胞传代到细胞密度3×105cells/mL,继续培养。
(5)当细胞密度增长到4×106cells/mL后,使用含有50μM MSX的CD OPM培养基将细胞扩增或传代,最低接种密度仍是3×105cells/mL,可以开始准备冻存或扩大培养。
(6)抗体收集方法:使用protein A亲和层析进行抗体纯化。
取外购Biolegend鼠抗人CD16[B73.1]10μg,实施例1制备得到的自制鼠抗人CD16[ZXCloneHCD16-04]10μg,自制兔抗人CD16[ZXCloneHCD16-05]10μg与上样缓冲液混合(抗体与上样缓冲液的体积比为4:1),100℃水浴30min,10000rpm室温离心1min后得到待测样品。取出预制胶安装于电泳槽内,注入电泳缓冲液,拨梳子,上样。其中,蛋白Marker上样10μL,待测样品上样5μg;电泳仪设置:电压80V,电泳时间90min,溴酚蓝指示带至底部时即可结束电泳;取出凝胶放于考马斯亮蓝染色30min,置于脱色液中脱色1h,重复5次后,拍照。
结果表明实施例1制备得到的抗人CD16抗体重轻链完整,条带清晰,纯度达到95%以上,结果见图1。
实施例2抗体标记
1、FITC标记
(1)分别取自制鼠抗人CD16[ZXCloneHCD16-04]0.2mg和自制兔抗人CD16[ZXCloneHCD16-05]0.2mg各于超滤管内,用浓度1mM的PBS洗涤抗体3次后。4000g,2min倒立离心超滤管,收集浓缩抗体,用浓度为1mM的PBS稀释至10mg/mL。
(2)取小分子染料FITC(按n(摩尔质量)抗体:n(摩尔质量)FITC=1:15加染料)加入单克隆抗体反应液中,混匀。
(3)25℃室温,于振荡器上避光反应45min。
(4)转移反应液于超滤离心管内,用浓度1mM的PBS洗涤抗体5次后。4000g,2min倒立离心超滤管,收集浓缩抗体,用浓度1mM的PBS稀释抗体终浓度为0.5mg/mL。
标记后的抗体记为自制鼠抗人CD16[ZXCloneHCD16-04]-FITC和自制兔抗人CD16[ZXCloneHCD16-05]-FITC。
2、APC标记
按照“荧光蛋白和/或偶联蛋白单克隆抗体标记方法及其试剂盒(CN202010972671.X)”专利所述方法分别标记自制鼠抗人
CD16[ZXCloneHCD16-04]和自制兔抗人CD16[ZXCloneHCD16-05],标记后的抗体记为自制鼠抗人CD16[ZXCloneHCD16-04]-APC和自制兔抗人CD16[ZXCloneHCD16-05]-APC。
3、标记荧光抗体效果检测
(1)于1.5mL离心管中加入100μL细胞质控品(厂家:Beckman Coulter,货号:6607077)。
(2)取1μL的荧光标记抗体自制鼠抗人CD16[ZXCloneHCD16-04]-FITC、自制兔抗人CD16[ZXCloneHCD16-05]-FITC、自制鼠抗人
CD16[ZXCloneHCD16-04]-APC和自制兔抗人CD16[ZXCloneHCD16-05]-APC分别加入样本中;取1μL的对照荧光抗体外购Biolegend鼠抗人CD16[B73.1]-FITC(厂家:Biolegend,货号:360702)、外购Biolegend鼠抗人CD16[B73.1]-APC(厂家:Biolegend,货号:360702)分别加入样本中。
(3)上述样品均避光反应15分钟。
(4)加入1mL溶血素,继续反应15分钟,溶血素选用正熙流式细胞仪用溶血素(溶血素:浙湖械备20200133号)。
(5)4000rpm离心2min,弃上清,加入500μL PBS重悬待检测样本。
(6)流式细胞仪(贝克曼DxFlex流式细胞仪)检测样本阳性细胞检测比例、图谱分群情况等指标,进而确定自制鼠抗人CD16[ZXCloneHCD16-04]和自制兔抗人CD16[ZXCloneHCD16-05]是否能标记上荧光染料FITC和APC,以及标记的荧光抗体检测靶抗原的是否正确。
FITC和APC均成功标记自制鼠抗人CD16[ZXCloneHCD16-04]和自制兔抗人CD16[ZXCloneHCD16-05],与外购Biolegend鼠抗人CD16[B73.1]的比例相近,见图2-图7。
实施例3抗体效价
抗体效价检测方法:
本实施例选用抗体为:
抗体1:阳性对照,外购Biolegend鼠抗人CD16[B73.1](厂家:Biolegend,货号:360702);
抗体2:自制鼠抗人CD16[ZXCloneHCD16-04];
抗体3:自制兔抗人CD16[ZXCloneHCD16-05]。
首先,将已知浓度抗体1,抗体2,抗体3先稀释为1mg/mL,然后用PBS按1:100,1:1000,1:3000,1:6000,1:12000,1:24000,1:48000,1:60000,1:96000比例再次对抗体进行稀释。抗体2和抗体3作为实验组,抗体1作为对照组。
实验组为两组,对照组一组,每组取100μL健康人血企业参考品,向每组中加入1μLCD16-APC商品化流式荧光抗体,避光孵育15分钟后,向3组实验中分别加入1mL溶血素(正熙流式细胞仪用溶血素溶血素:浙湖械备20200133号),继续反应15min。4000rpm离心2min,弃上清。
实验组分别加入按比例稀释的100μL抗体2、抗体3,对照组分别加入按比例稀释的100μL抗体1。混合后室温避光反应30min,加入900μL PBS重悬混匀样本,4000rpm离心2min,弃上清,再加入500μL PBS重悬待检测样本,流式细胞仪检测。
3种抗体的亲和力分别为:
抗体1:外购Biolegend鼠抗人CD16[B73.1],其亲和力(kd)为1324.6(1mg/mL);
抗体2:自制鼠抗人CD16[ZXCloneHCD16-04],其亲和力(kd)为;395.4(1mg/mL);
抗体3:自制兔抗人CD16[ZXCloneHCD16-05],其亲和力(kd)为405.2(1mg/mL)。
结果表明自制鼠抗人CD16[ZXCloneHCD16-04]与自制兔抗人CD16[ZXCloneHCD16-05]亲和力均强于外购Biolegend鼠抗人CD16[B73.1]。
实施例4抗体特异性
本实施例选用抗体为:
抗体(1):实施例1制备得到的抗人CD16抗体[ZXCloneHCD16-04];
抗体(2):实施例1制备得到的抗人CD16抗体[ZXCloneHCD16-05]。
商品外购的纯抗:
抗体(3):外购Biolegend鼠抗人CD16[B73.1](厂家:Biolegend,货号:360702)
用于检测的商品化外购的荧光抗体:
荧光抗体(4):外购Biolegend鼠抗人CD16[B73.1]-FITC(厂家:Biolegend,货号:360702)。
用于共染的商品化外购的荧光抗体:
荧光抗体(5):CD3-APC(厂家:隆洋正熙生物技术有限公司,货号:110610272)。
抗体特异性检测方法:
于1.5mL离心管,分别加入100μL细胞质控品(厂家:Beckman Coulter,货号:6607077)和1mL溶血素,反应15分钟。溶血素选用正熙流式细胞仪用溶血素溶血素:浙湖械备20200133号)。4000rpm,离心2min,弃上清,加入100μL PBS重悬细胞。
实验分为纯抗检验组和阳性对照组。
实验组:分别加入抗人CD16抗体[ZXCloneHCD16-04]和CD16抗体[ZXCloneHCD16-05]3μL,避光反应15分钟后,加入500μL PBS重悬样本,4000rpm离心2min,弃上清,再加入100μL PBS重悬样本。在2个经纯抗CD16[ZXCloneHCD16-04]封闭后样本中分别加入
CD16[ZXCloneHCD16-04]-FITC。在2个经纯抗CD16[ZXCloneHCD16-05]封闭后样本中分别加入CD16[ZXCloneHCD16-05]-FITC。
对照组:在重悬样本分别加入CD16[ZXCloneHCD16-04]-FITC 1μL;在重悬样本分别加入CD16[ZXCloneHCD16-05]-FITC 1μL。
对照组和实验组均加入共染抗体CD3-APC 1μL。
所有样本避光反应15分钟后。加入500μL PBS重悬样本,4000rpm离心2min,弃上清,再加入500μL PBS重悬样本。
流式细胞仪(安捷伦NovoCyte流式细胞仪)检测样本阳性细胞比例、图谱分群情况等指标,进而确定ZXCloneHCD16-04和ZXCloneHCD16-05是否能特异性识别CD16靶抗原。
由图11-图14可知,实验组则有CD3-APC信号,无CD16[ZXCloneHCD16-04]-FITC和CD16[ZXCloneHCD16-05]-FITC荧光抗体的信号。对照组,均有CD3和CD16荧光抗体的信号。以此说明,由CHO/HEK293制备所得的CD16抗体能特异性识别CD16靶抗原。
Claims (10)
1.一种抗CD16抗体,其特征在于,包括重链和轻链,所述的重链包括重链可变区,所述的轻链包括轻链可变区;所述的重链可变区包括HCDR1、HCDR2和HCDR3,所述的轻链可变区包括LCDR1、LCDR2和LCDR3,其中:
(1)HCDR1为SEQ ID NO.1所示的氨基酸序列;
(2)HCDR2为SEQ ID NO.2所示的氨基酸序列;
(3)HCDR3为SEQ ID NO.3所示的氨基酸序列;
(4)LCDR1为SEQ ID NO.4所示的氨基酸序列;
(5)LCDR2为SEQ ID NO.5所示的氨基酸序列;
(6)LCDR3为SEQ ID NO.6所示的氨基酸序列。
2.根据权利要求1所述的抗CD16抗体,其特征在于,所述的抗CD16抗体重链可变区的氨基酸序列为SEQ ID NO.7或与SEQ ID NO.7具有80%以上序列相似性的序列;所述的抗CD16抗体轻链可变区的氨基为SEQ ID NO.8或与SEQ ID NO.8具有80%以上序列相似性的序列;所述的抗CD16抗体轻链可变区的氨基为SEQ ID NO.13或与SEQ ID NO.13具有80%以上序列相似性的序列。
3.根据权利要求1所述的抗CD16抗体,其特征在于,所述的抗CD16抗体重链氨基酸序列为SEQ ID NO.9,或与SEQ ID NO.9具有65%以上序列相似性的序列;
或所述的抗CD16抗体重链氨基酸序列为SEQ ID NO.11,或与SEQ ID NO.11具有65%以上序列相似性的序列;
所述的抗CD16抗体轻链氨基酸序列为SEQ ID NO.10,或与SEQ ID NO.10具有65%以上序列相似性的序列;
或所述的抗CD16抗体轻链氨基酸序列为SEQ ID NO.12,或与SEQ ID NO.12具有65%以上序列相似性的序列。
4.根据权利要求1-3任一项所述的抗CD16抗体,其特征在于,为单克隆抗体。
5.根据权利要求4所述的抗CD16抗体,其特征在于,为鼠源化抗体或兔源化抗体。
6.表达权利要求1-5任一项所述的抗CD16抗体的核苷酸序列;优选地,重链SEQ IDNO.9和SEQ ID NO.11对应的核苷酸序列分别为SEQ ID NO.14和SEQ ID NO.15所示、轻链SEQ ID NO.10和SEQ ID NO.12对应的核苷酸序列分别为SEQ ID NO.16和SEQ ID NO.17所示。
7.包括权利要求6所述的核苷酸序列的表达载体。
8.包括权利要求1-5任一项所述的抗CD16抗体或权利要求6所述的核苷酸序列或权利要求7所述的表达载体的细胞。
9.一种CD16检测方法,其特征在于,通过权利要求1-5任一项所述的抗CD16抗体进行检测,所述的方法为非疾病诊断或治疗方法。
10.一种CD16检测试剂盒,其特征在于,包括权利要求1-5任一项所述的抗CD16抗体或权利要求6所述的核苷酸序列或权利要求7所述的表达载体或权利要求8所述的细胞。
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