CN116554337A - 针对小鼠免疫球蛋白G3亚型（IgG3）兔单克隆抗体及其应用 - Google Patents
针对小鼠免疫球蛋白G3亚型（IgG3）兔单克隆抗体及其应用 Download PDFInfo
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- CN116554337A CN116554337A CN202210597752.5A CN202210597752A CN116554337A CN 116554337 A CN116554337 A CN 116554337A CN 202210597752 A CN202210597752 A CN 202210597752A CN 116554337 A CN116554337 A CN 116554337A
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Abstract
本发明涉及生物技术领域，公开了兔抗小鼠免疫球蛋白G3亚型(IgG3)的特异性单克隆抗体OTIR8B8，所述的抗体是利用分选特异B细胞并培养筛选及分子克隆重组产生。所述兔单克隆抗体OTIR8B8的免疫原为天然小鼠IgG3(mIgG3)抗体，其OTIR8B8抗体轻链(VL)的氨基酸序列如SEQIDNO.1所示；OTIR8B8抗体的重链(VH)的氨基酸序列如SEQIDNo.2所示。所述OTIR8B8抗体的VL包括3个抗原决定簇：CDR1、CDR2和CDR3，其氨基酸序列分别如SEQIDNo.3‑5所示，所述OTIR8B8抗体的VH区域包括3个抗原决定簇：CDR1、CDR2和CDR3，其氨基酸序列分别如SEQIDNo.6‑8所示。本发明还涉及抗小鼠免疫球蛋白G3亚型(IgG3)特异性兔单克隆抗体在免疫检测工具中的应用，包含但不限于用于一抗的化学发光、荧光和显色检测，适合各种应用，例如细胞成像、流式细胞检测、蛋白质免疫印迹及免疫组织化学，也为下一步工程抗体的制备提供基础。
Description
技术领域
本发明属于生物技术领域，尤其涉及抗小鼠免疫球蛋白G3亚型(IgG3)兔单克隆抗体及其在免疫检测方面应用。
背景技术
亲和结合物/抗体是生命科学中最常用的工具之一，用于研究蛋白以及它们在各种生物通路和疾病中的功能。目前在科学研究领域及医药领域抗体种属应用最广泛的包括鼠源抗体、人源抗体、兔源抗体。这些抗体可以特异性识别兴趣靶标，然后通过酶标二抗或荧光素标记二抗显示信号。目前抗鼠IgG及抗人IgG标记抗体市场应用十分之广，可应用于酶联免疫吸附(ELISA)、免疫印迹 (Westernblot)、免疫组织化学染色(IHC)、流式细胞术(FCM)及免疫共沉淀等，最常用的标记方法包括辣根过氧化物酶(HRP)，碱性磷酸酶，生物素，荧光素等。
目前制备抗鼠IgG抗体的主要方法是将IgG免疫球蛋白直接免疫兔或羊或驴等动物产生免疫学清多克隆抗体，然后纯化后应用。但是研发IgG重链和轻链不同类型特异性抗体，多克隆抗体是远远不够的，需要制备单克隆抗体，特别是针对不同亚型mIgG的单克隆抗体。
鼠IgG单克隆抗体重链包括IgG1,IgG2a,IgG2b,IgG2c,IgG3五种亚型，轻链包括κ链及λ链两种类型，κ链：λ链约为20：1。目前市场上大都是多克隆抗体，如山羊多抗、绵羊多抗、驴多抗等，很少有单克隆抗体，特别是没有兔抗mIgG3 特异性单克隆抗体。
在研发抗mIgG3抗体时，因为要获得特异性识别mIgG3，而不与mIgG1, mIgG2a,mIgG2b,mIgG2c,humanIgG1(hIgG1),humanIgG2(hIgG2),humanIgG3(hIgG 3),humanIgG4(hIgG4),RatIgG,Rabbit IgG(RabIgG)有交叉反应的单克隆抗体，我们遇到了很多困难。在此期间我们经过多次分选优化及大量筛选最终获得了本发明申请兔单克隆抗体OTIR8B8。
特异性识别抗mIgG3亚型的抗体，包括裸抗及标记抗体市场应用十分之广，可应用于酶联免疫吸附(ELISA)、免疫印迹(Westernblot)、免疫组织化学染色 (IHC)、流式细胞术(FCM)及免疫共沉淀等。然而以上应用实验的核心为特异性结合mIgG3的单克隆抗体，其性能的优劣直接决定着整个检测的灵敏度和特异性。因此，寻找高特异性和高敏感性的抗mIgG3的抗体是建立检测mIgG3 方法的关键。检索中国、美国、EPO、WIPO等专利网站，利用mIgG3或 mIgG3+monoclonal antibody或antibody作为关键词，有关与mIgG3具有高亲和力和特异性结合的单克隆抗体的专利没有查到，有关检测利用抗mIgG3兔单克隆抗体OTIR8B8制备免疫检测试剂盒也未查到；利用mIgG3或 mIgG3+monoclonal antibody或antibody作为关键词，在pubmed未查到与本发明申报内容相关的类似文献。查询国家药品监督管理网站，还没有与mIgG3相关的检测试剂注册。
发明内容
本发明的目的是提供特异性好、亲和力高的抗mIgG3兔单克隆抗体 OTIR8B8及其在免疫检测工具中的应用，包含但不限于用于一抗的化学发光、荧光和显色检测，适合各种应用，例如细胞成像、流式细胞检测、蛋白质免疫印迹及免疫组织化学，也为下一步工程抗体的制备提供基础。
所述兔单克隆抗体为兔单克隆抗体OTIR8B8。
所述兔单克隆抗体OTIR8B8以天然mIgG3抗体为免疫原，通过免疫注射新西兰大白兔，取免疫兔子外周血单核细胞(PMBCs)，分选特异性B细胞培养、筛选并利用分子克隆重组技术获得。
所述兔单克隆抗体OTIR8B8，其轻链可变区(VL)含108aa，其氨基酸序列如SEQ IDNO.1所示；所述抗体的重链(VH)含109aa，其氨基酸序列如SEQ IDNo.2所示。
所述兔单克隆抗体OTIR8B8，其中VL区域包括3个抗原决定簇：CDR1、 CDR2和CDR3，抗原决定簇的区域相应地分别为27aa-34aa，52aa-54aa和 91aa-98aa。其氨基酸序列分别如SEQ ID No.3-5所示。
所述兔单克隆抗体OTIR8B8，其中VH区域包括3个抗原决定簇：CDR1、 CDR2和CDR3，抗原决定簇的区域相应地分别为25aa-32aa，50aa-56aa和 93aa-98aa。其氨基酸残基序列分别如SEQ ID No.6-8所示。
所述的兔单克隆抗体OTIR8B8可以与mIgG3高特异性结合，可通过本领域技术人员所公知的的方法，做为一抗或各种标记二抗使用。尤其是，做为酶标二抗或荧光素标记二抗应用于免疫组化检测、免疫印迹检测及流式细胞检测。
附图说明
图1为兔单克隆抗体OTIR8B8重链和轻链全长扩增产物的电泳图，M为 DNA分子量Marker；
图2为兔单克隆抗体OTIR8B8特异识别天然mIgG3抗体的Westernblot检测结果图；
图3为兔单克隆抗体OTIR8B8特异识别mIgG3抗体线性ELISA检测结果图；
图4为兔单克隆抗体OTIR8B8与其它种属IgG交叉反应ELISA检测结果图；
图5为辣根过氧化物酶标记兔单克隆抗体OTIR8B8特异识别天然mIgG3 抗体的Westernblot检测结果图；
图6为辣根过氧化物酶标记兔单克隆抗体OTIR8B8特异识别mIgG3抗体的 ELISA检测结果图。
具体实施方式
下面结合具体实施例，进一步阐述本发明。应理解，这些实施例仅用于说明本发明而不用于限制本发明的范围。下列实施例中未注明具体条件的实验方法,通常按照常规条件、实验室手册中所述的条件或按照制造厂商所建议的条件。
实施例1:mIgG3兔单克隆抗体的制备
一、mIgG3免疫原的制备
采购Rockland公司mIgG3抗体做为免疫原
二、动物免疫
上述采购的mIgG3抗体以完全弗氏佐剂乳化，采用皮下注射方法免疫2kg 左右的新西兰大白兔，免疫剂量为500μg/只，间隔两周后进行第二次免疫，以不完全弗氏佐剂乳化，免疫剂量为250μg/只。免疫两次后取尾血以ELISA法梯度稀释测定血清效价；依据ELISA效价128000时的OD450大于1.0为标准，根据结果判定是否收集PBMCs或是继续免疫，选取抗体效价最高的兔子进行PBMCs收集。
三、PBMCs分离、特异性B细胞分选、克隆重组将兔仰卧固定在手术台上，将心脏部位被毛减掉，用酒精消毒皮肤，选择心搏最明显处用50ml注射器穿刺，针头刺入心脏后即有血液涌入注射器，取得所需血量后迅速将针头拔出，将注射器中的全血转入无菌50ml管中，与等量的PBS混匀后逐滴缓慢的加入到淋巴细胞分离液上方，室温400×g离心30分钟，离心后，液面由上至下分为四层：黄色血浆层、白色薄膜层即单个核细胞层、分离液层及红细胞层，小心吸取单个核细胞层并用PBS洗涤去除血小板和淋巴细胞分离液后即可获得兔PBMCs。从兔PBMCs中继续分选抗原特异性的B细胞进行培养，培养后的B 细胞上清用抗原包被的ELISA板筛选阳性克隆。阳性克隆的细胞收集裂解后提取RNA并反转录成cDNA,天然配对的兔单克隆抗体轻重链全长序列从对应阳性克隆的cDNA中被扩增出来，通过克隆重组方法构建兔单克隆抗体表达载体，并经测序确定序列。扩增的全长PCR产物结果如图1。
四、单克隆抗体的制备和纯化为了获得识别人mIgG3蛋白的兔单克隆抗体，本发明将兔单克隆抗体重链、轻链基因装载在表达载体上，将质粒转染HEK293 细胞；转染120-144小时获得培养上清中含有重组的识别人mIgG3蛋白的兔单克隆抗体。收取细胞悬液，离心取上清，亲和层析法进行抗体纯化。以BCA法测定纯化后的单抗浓度，再分装、冻干。
实施例2:抗mIgG3兔单克隆抗体OTIR8B8的鉴定
一、兔单克隆抗体OTIR8B8 Westernblot鉴定
采用Westernblot(WB)检测。mIgG1、mIgG2a、mIgG2b、mIgG3每个亚型各选择2个不同样品，每个蛋白上样100ng进行SDS-PAGE，转膜后进行WB 检测。
结果显示，兔单克隆抗体OTIR8B8能特异识别mIgG3，不识别mIgG1、 mIgG2a、mIgG2b，抗体稀释比：1:5000-10000,1mg/ml，结果见图2。
二、兔单克隆抗体OTIR8B8 ELISA鉴定
mIgG1、mIgG2a、mIgG2b、mIgG3、mIgG3 Fc、mIgG3 F(ab')2抗体包被酶标板，4℃过夜；第二天取出酶标板，PBST洗涤一次，1％BSA溶液37℃封闭2 小时，PBST洗涤3次；每孔加入兔单克隆抗体OTIR8B8100μl，浓度分别为10、 3、1、0.3、0ng/ml，37℃孵育1小时；孵育完成后取出酶标板，PBST洗涤3 次，分别加入HRP标记的羊抗兔二抗作为检测抗体，37℃孵育1小时；孵育完成后取出酶标板，PBST洗涤5次，加入TMB底物，37℃显色10min。取出后加终止液，在酶标仪上测定OD450读数。采用同样方法检测兔单克隆抗体 OTIR8B8与其它种属IgG交叉识别。
结果显示，兔单克隆抗体OTIR8B8能特异识别mIgG3，且识别的是Fc片段，不识别mIgG1、mIgG2a、mIgG2b，效价可达到1.5*107，结果见图3。图4 结果说明兔单克隆抗体OTIR8B8能特异识别mIgG3，不与其它种属IgG交叉识别，包括human IgG(hIgG1、hIgG2、hIgG3、hIgG4),RatIgG,RabIgG。
实施例3:兔单克隆抗体OTIR8B8可变区基因与氨基酸序列分析
以OTIR8B8抗体的重组质粒为DNA模板，根据模板上轻链及重链5'端载体序列设计轻链可变区及重链可变区测序引物，采用测序仪ABI 3730进行测序。经测序获得兔单克隆抗体OTIR8B8轻链及重链可变区的核苷酸序列。
利用互联网，在http://www.imgt.org上使用IMGT/V-QUEST分析软件，分别将轻链与重链的核苷酸序列进行测序结果数据分析，得到兔单克隆抗体 OTIR8B8的轻链氨基酸序列如SEQ ID NO.1所示，重链氨基酸序列如 SEQ ID NO.2所示。VL全长为108个氨基酸，其FR的4个结构域氨基酸数分别为26、17、36和10，CDR的3个结构域氨基酸数分别为8、3和8，CDR1、 CDR2和CDR3的区域相应地分别为27aa-34aa，52aa-54aa和91aa-98aa，其氨基酸序列分别为QSVYNNNW、KAS、QGTYSGGT。
通过分析，所述兔单克隆抗体OTIR8B8 VH全长为109个氨基酸，其FR 的4个结构域氨基酸数分别为24、17、36和11，CDR的3个结构域氨基酸数分别为8、7和6，CDR1、CDR2和CDR3分别为25aa-32aa，50aa-56aa和93aa-98aa，其氨基酸序列分别为GFSLSSYA、INAGNRP、SRGDNL。
实施例4:HRP标记兔单克隆抗体OTIR8B8作为二抗的鉴定
一、HRP标记兔单克隆抗体OTIR8B8：
1、兔单克隆抗体OTIR8B8溶于PH9.6的碳酸氢钠溶液中；
2、取一定质量的HRP溶于去离子水中，加入高碘酸钠反应30min，加入乙二醇继续反应30min，过夜透析；
3、称取一定量的硼氢化钠溶于去离子水中，向交联完毕的抗体-HRP溶液中加入硼氢化钠，反应2h，过夜透析；
4、将获得的HRP标记抗体加入等量的甘油-20摄氏度保存。
二、鉴定：
1.HRP标记兔单克隆抗体OTIR8B8 Westernblot鉴定
采用Western blot(WB)检测。取mIgG3抗体100ng进行SDS-PAGE，转膜后孵育HRP标记的兔单克隆抗体OTIR8B8进行WB检测。
结果显示，HRP标记的兔单克隆抗体OTIR8B8能很好的识别mIgG3 Fc 区域，分子量50KD左右，抗体稀释比：1:5000-10000,1mg/ml，结果见图5。
三、兔单克隆抗体OTIR8B8 ELISA鉴定
羊抗鼠IgG包被酶标板，4℃过夜；第二天取出酶标板，PBST洗涤一次， 1％BSA溶液37℃封闭2小时，PBST洗涤3次；每孔加入mIgG3抗体100ul，浓度为0.2ug/ml，37℃孵育1小时；孵育完成后取出酶标板，PBST洗涤3次，加入HRP标记的OTIR8B8作为检测抗体，1ug/ml开始倍比稀释7个梯度，37℃孵育1小时；孵育完成后取出酶标板，PBST洗涤5次，加入TMB底物，37℃显色10min。取出后加终止液，在酶标仪上测定OD450读数。
结果显示，HRP标记的兔抗mIgG3 OTIR8B8作为二抗能很好的检测 mIgG3抗体信号，1mg/ml时稀释比可以达到36K-18K，优于商品化羊抗鼠IgG 多克隆抗体(作为二抗可同时检测mIgG1、mIgG2a、mIgG2b、mIgG3)，结果见图6。
序列表
<110> 无锡傲锐东源生物科技有限公司
<120> 针对小鼠免疫球蛋白G3亚型（IgG3）兔单克隆抗体及其应用
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Claims (7)
1.抗小鼠免疫球蛋白G3亚型(IgG3)的特异性兔单克隆抗体，其特征在于：所述兔单克隆抗体为兔单克隆抗体OTIR8B8。
2.根据权利要求1所述的抗小鼠免疫球蛋白G3亚型(IgG3)特异性兔单克隆抗体OTIR8B8，其特征在于：兔单克隆抗体OTIR8B8以天然mIgG3抗体为免疫原，通过免疫注射新西兰大白兔，取免疫兔子外周血单核细胞(PMBCs)，分选特异性B细胞培养、筛选并利用分子克隆重组技术获得。
3.根据权利要求2所述的抗小鼠免疫球蛋白G3亚型(IgG3)特异性兔单克隆抗体OTIR8B8，其特征在于：特异性识别Fcγ区域。
4.根据权利要求2所述的抗小鼠免疫球蛋白G3亚型(IgG3)特异性兔单克隆抗体OTIR8B8，其特征在于:其轻链可变区(VL)含108aa，其氨基酸序列如SEQ ID NO.1所示；所述抗体的重链(VH)含109aa，其氨基酸序列如SEQ ID No.2所示。
5.根据权利要求2所述的抗小鼠免疫球蛋白G3亚型(IgG3)特异性兔单克隆抗体OTIR8B8，其中VL区域包括3个抗原决定簇：CDR1、CDR2和CDR3，抗原决定簇的区域相应地分别为27aa-34aa，52aa-54aa和91aa-98aa。其氨基酸序列分别如SEQ ID No.3-5所示。
6.根据权利要求2所述的抗小鼠免疫球蛋白G3亚型(IgG3)特异性兔单克隆抗体OTIR8B8，其中VH区域包括3个抗原决定簇：CDR1、CDR2和CDR3，抗原决定簇的区域相应地分别为25aa-32aa，50aa-56aa和93aa-98aa。其氨基酸残基序列分别如SEQ ID No.6-8所示。
7.权利要求2所述的抗小鼠免疫球蛋白G3亚型(IgG3)特异性兔单克隆抗体的应用，其特征在于:能特异性识别小鼠免疫球蛋白G3亚型(IgG3)，不交叉识别小鼠免疫球蛋白G1亚型(IgG1)、小鼠免疫球蛋白G2a亚型(IgG2a)、小鼠免疫球蛋白G2b亚型(IgG2b)；用于小鼠免疫球蛋白G3亚型(IgG3)在免疫检测工具中的应用，包含但不限于用于一抗的化学发光、荧光和显色检测，例如细胞成像、流式细胞检测、蛋白质免疫印迹及免疫组织化学等应用。
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