CN114276455A - 抗HIgG-Fc单克隆抗体及其制备方法与应用 - Google Patents
抗HIgG-Fc单克隆抗体及其制备方法与应用 Download PDFInfo
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- CN114276455A CN114276455A CN202111577260.1A CN202111577260A CN114276455A CN 114276455 A CN114276455 A CN 114276455A CN 202111577260 A CN202111577260 A CN 202111577260A CN 114276455 A CN114276455 A CN 114276455A
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Abstract
本发明提供了一种抗HIgG‑Fc单克隆抗体及其制备方法与应用，所述抗体包含有重链可变区的抗原互补决定区aaCDR1、aaCDR2和aaCDR3，轻链可变区的抗原互补决定区aaCDR1、aaCDR2和aaCDR3。提供了一种抗HIgG‑Fc单克隆抗体，并提供了相应的制备方法，本发明的所述抗HIgG‑Fc单克隆抗体可以应用于在制备生物药物中的研究，为药物的分析和药物性质的解释提供了基础。
Description
技术领域
本发明属于生物医药技术领域，具体涉及一种抗HIgG-Fc单克隆抗体及其制备方法与应用。
背景技术
免疫球蛋白是指具有抗体（Ab）活性或化学结构，与抗体分子相似的球蛋白。其主要为IgG，在许多病理情况下，人体免疫系统会产生变化，通过检测血液IgG的变化可以指导疾病的诊断。随着生物医药的快速发展，抗体药占居很大的一部分，从早期的鼠源抗体到人鼠嵌合抗体，再到目前主要以人源化抗体和全人抗体为主的时代，需要不同方法来分析和解释药物的一些性质。如何制备一种Anti- Human IgG Fc protein单克隆抗体，以更好的为生物药物的开发及临床检测分析提供些实用工具成为了目前待解决的问题。
发明内容
为了解决现有技术的不足，本发明提供了一种抗HIgG-Fc单克隆抗体及其制备与应用。
本发明的目的通过以下技术方案来实现：
抗HIgG-Fc单克隆抗体，包含有重链可变区的抗原互补决定区aaCDR1、aaCDR2和aaCDR3，其氨基酸序列分别为SEQ ID NO:1、SEQ ID NO:2、SEQ ID NO:3；
轻链可变区的抗原互补决定区aaCDR1、aaCDR2和aaCDR3，其氨基酸序列分别为SEQID NO:4、SEQ ID NO:5、SEQ ID NO:6。
优选地，所述抗体具有SEQ ID NO：7所示的重链区可变区氨基酸序列和SEQ IDNO:8所示的轻链区可变区氨基酸序列。
一种核酸分子，所述核酸分子包括编码以上任一所述的抗HIgG-Fc单克隆抗体。
一种细胞系，能够产生如以上任一所述的抗HIgG-Fc单克隆抗体。
抗HIgG-Fc单克隆抗体的制备方法，所述方法包括，培养如以上所述的细胞系，并产生所述的抗HIgG-Fc单克隆抗体。
抗HIgG-Fc单克隆抗体的应用，所述抗HIgG-Fc单克隆抗体在生物药品的开发中的应用。
本发明的有益效果体现在：提供了一种抗HIgG-Fc单克隆抗体，并提供了相应的制备方法，本发明的所述抗HIgG-Fc单克隆抗体可以应用于在制备生物药物中的研究，为药物的分析和药物性质的解析提供了基础。
附图说明
图1：本发明实施例5中抗体在450 nm波长下的OD值。
具体实施方式
本发明揭示了一种抗HIgG-Fc单克隆抗体，包含有重链可变区的抗原互补决定区aaCDR1、aaCDR2和aaCDR3，其氨基酸序列分别为SEQ ID NO:1 GFTFSNYA、SEQ ID NO:2ISTDSGTT、SEQ ID NO:3 ARQGYPHWYFDV；轻链可变区的抗原互补决定区aaCDR1、aaCDR2和aaCDR3，其氨基酸序列分别为SEQ ID NO:4 QDIYNY、SEQ ID NO:5 YTS、SEQ ID NO:6LQYDYLLFT。
所述抗体具有SEQ ID NO：7所示的重链区可变区氨基酸序列和SEQ ID NO:8所示的轻链区可变区氨基酸序列。
其中，SEQ ID NO：7：
EMKLVESGGGLVQPGGSLKLSCAASGFTFSNYAMSWVRQTPEKRLEWVAYISTDSGTTYYPDTVKGRFTISRDNAKNTLYLHMSSLKSEDTAMYFCARQGYPHWYFDVWGAGTTVTVSS
SEQ ID NO:8：
DIQMTQSPSSLSASLGGKVTITCKASQDIYNYIAWYQDKPGKGPRLLIHYTSTLQPGIPSRFSGSGSGRDYSFSISNLEPDDVATYYCLQYDYLLFTFGSGTKLEIK
本发明还揭示了一种核酸分子及细胞系，以及以上所述抗HIgG-Fc单克隆抗体的制备方法。
所述核酸分子由编码以上任一所述的抗HIgG-Fc单克隆抗体获得。所述细胞系能够产生如以上任一所述的抗HIgG-Fc单克隆抗体。所述方法包括培养上述的细胞系，并产生所述的抗HIgG-Fc单克隆抗体。本发明的所述抗HIgG-Fc单克隆抗体可以应用于在制备生物药物中的研究。
为更好的理解本发明，以下进行具体的实施举例说明。
实施例1：抗原准备
准备Human IgG Fc protein（Fitzgerald公司），用分子筛将保存液转换到磷酸盐缓冲体系，加入弗氏完全佐剂（sigma）备用。
实施例2：动物免疫
将实施例1中准备的抗原（Human IgG Fc protein）用银汞调合器进行混匀乳化，免疫4~6周龄雌性Balb/c小鼠（购自北京维通利华实验动物技术有限公司），皮下多点注射，剂量为85µg/只。每14天加强免疫一次，抗原使用弗氏非完全佐剂乳化，剂量为50µg/只。第3次加强免疫后7天，以间接ELISA（波长450nm）检测小鼠血清中抗免疫原的多抗效价，效价最高的小鼠以尾静脉注射冲击免疫，抗原用生理盐水混匀，剂量为50µg/只。
实施例3：杂交瘤细胞制备
1、融合：将实施例2中免疫达标的小鼠采血，取脾脏制备细胞悬液，与小鼠骨髓瘤细胞Fo以10：1比例混合，离心1200rpm，5分钟。弃上清后，离心管放入37℃水浴中，在1分钟内缓慢加入1 mL的PEG1500（Roche公司），并搅动细胞。在温水中静置l分钟后，加入10 mL无血清的IMDM（Hyclone公司），混匀，离心1000rpm，5分钟。弃上清后，加入含有10 %胎牛血清（Excell公司）的培养基，将细胞吹打起来，并加入配好的HAT完全培养基（Sigma公司）混匀，铺96孔板。将铺好细胞的培养板，放入37℃、5% CO2培养箱中培养。
2、筛选及亚克隆：利用ELISA的方法进行初步的筛选，将筛选得到的阳性抗体进行稀释直至得到单个亚克隆细胞。通过ELISA筛选，得到多个阳性克隆，挑选其中一个阳性克隆进行扩大培养，检测定株。以下以编号02CT6.2.1为挑选的个体举例说明。
实施例4：腹水诱生法制备单克隆抗体
1、腹水制备
对数生长期细胞用无血清培养基洗涤并悬起，计数3-8x105/mL，1mL悬浮的细胞腹腔注射事先用石蜡油致敏的小鼠。7天后开始收集腹水。取出的腹水于4℃下离心4000rpm，10分钟。小心吸出中间的腹水收集于离心管中，4℃或-20℃保存。
2、单克隆抗体纯化
用Protein G（GE公司）亲和层析法按说明书从腹水中纯化抗体。SDS-PAGE胶鉴定纯度，Bradford法测定浓度。纯化的抗体保存于-20℃。
实施例5：单克隆抗体特性鉴定
1、亚型检测：选用Mouse Immunoglobulin Isotyping ELISA试剂盒（BD），按操作说明，羊抗鼠IgG板子包被、封闭。取杂交瘤上清，进行检测。结果显示，克隆02CT6.2.1为IgG1 λ型鼠源单克隆抗体。
2.抗体效价检测
用PBS（pH 7.2）稀释包被Human IgG Fc protein，1µg/mL，每孔加100 µL，4℃，过夜。倾空液体，用含0.05% Tween的PBS洗3次，每孔加入200 µL封闭液（含3% BSA的PBS），37℃下孵育1小时，倾空液体，备用。
每孔加入稀释好的抗体溶液，1:3做梯度稀释，每孔100 µL反应液，37℃下孵育1小时，倾空液体，用PBST清洗3次。用封闭液1:10000稀释HRP标记的羊抗鼠抗体Anti-MouseIgG(Fc specific)（sigma公司），每孔分别加入100 µL，37℃下孵育1小时，倾空液体，用PBST清洗4次。每孔加100µLTMB显色液进行显色反应，10-20分钟内测定450 nm波长下的OD值。结合图1所示，显示挑选的单抗具有与Human IgG Fc protein较好的结合活性。
实施例6：抗体可变区序列测定
取培养的杂交瘤细胞系1x106，离心取上清，将细胞沉淀委托苏州金唯智生物科技有限公司进行抗体重链和轻链可变区序列测定，如下示出示例性的抗体重链可变区序列和抗体轻链可变区序列。
抗体CDR氨基酸序列
| 样品编号 | 链型 | aaCDR1 | aaCDR2 | aaCDR3 |
| 02CT6.2.1-VH | IGH | GFTFSNYA | ISTDSGTT | ARQGYPHWYFDV |
| 02CT6.2.1-VL | IGK | QDIYNY | YTS | LQYDYLLFT |
抗体可变区氨基酸序列
重链可变区，SEQ ID NO:7
EMKLVESGGGLVQPGGSLKLSCAASGFTFSNYAMSWVRQTPEKRLEWVAYISTDSGTTYYPDTVKGRFTISRDNAKNTLYLHMSSLKSEDTAMYFCARQGYPHWYFDVWGAGTTVTVSS
轻链可变区，SEQ ID NO:8
DIQMTQSPSSLSASLGGKVTITCKASQDIYNYIAWYQDKPGKGPRLLIHYTSTLQPGIPSRFSGSGSGRDYSFSISNLEPDDVATYYCLQYDYLLFTFGSGTKLEIK
编码抗体可变区的核酸序列
重链可变区，SEQ ID NO:9
GAAATGAAGCTGGTGGAGTCTGGGGGAGGTTTAGTGCAGCCTGGAGGGTCCCTGAAACTCTCCTGTGCAGCCTCTGGATTCACTTTCAGTAACTATGCCATGTCTTGGGTTCGCCAGACTCCAGAGAAGAGGCTGGAGTGGGTCGCATATATTAGTACTGATAGTGGTACCACTTACTATCCAGACACTGTCAAGGGCCGATTCACCATCTCCAGAGACAATGCCAAGAACACCCTTTACCTGCATATGAGCAGTCTGAAGTCTGAAGACACGGCCATGTATTTCTGTGCAAGACAGGGGTATCCCCACTGGTACTTCGATGTCTGGGGCGCAGGGACCACGGTCACCGTCTCCTCA
轻链可变区，SEQ ID NO:10
GACATCCAGATGACACAGTCTCCATCCTCACTGTCTGCATCTCTGGGAGGCAAAGTCACCATCACTTGCAAGGCAAGCCAAGACATTTACAATTATATTGCTTGGTACCAAGACAAGCCTGGAAAAGGTCCTAGGCTGCTCATACATTACACATCTACATTACAGCCAGGCATCCCATCAAGGTTCAGTGGAAGTGGGTCTGGGAGAGATTATTCCTTCAGCATCAGCAACCTGGAGCCTGATGATGTTGCAACTTATTATTGTCTACAGTATGATTATCTTCTATTCACGTTCGGCTCGGGGACAAAGTTGGAAATAAAA。
且以上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精神和范围。
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Leu His Met Ser Ser Leu Lys Ser Glu Asp Thr Ala Met Tyr Phe Cys
85 90 95
Ala Arg Gln Gly Tyr Pro His Trp Tyr Phe Asp Val Trp Gly Ala Gly
100 105 110
Thr Thr Val Thr Val Ser Ser
115
<210> 8
<211> 107
<212> PRT
<213> 鼠源抗体(抗体可变区序列)
<400> 8
Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Leu Gly
1 5 10 15
Gly Lys Val Thr Ile Thr Cys Lys Ala Ser Gln Asp Ile Tyr Asn Tyr
20 25 30
Ile Ala Trp Tyr Gln Asp Lys Pro Gly Lys Gly Pro Arg Leu Leu Ile
35 40 45
His Tyr Thr Ser Thr Leu Gln Pro Gly Ile Pro Ser Arg Phe Ser Gly
50 55 60
Ser Gly Ser Gly Arg Asp Tyr Ser Phe Ser Ile Ser Asn Leu Glu Pro
65 70 75 80
Asp Asp Val Ala Thr Tyr Tyr Cys Leu Gln Tyr Asp Tyr Leu Leu Phe
85 90 95
Thr Phe Gly Ser Gly Thr Lys Leu Glu Ile Lys
100 105
Claims (6)
1.抗HIgG-Fc单克隆抗体，其特征在于：包含有重链可变区的抗原互补决定区aaCDR1、aaCDR2和aaCDR3，其氨基酸序列分别为SEQ ID NO:1、SEQ ID NO:2、SEQ ID NO:3；
轻链可变区的抗原互补决定区aaCDR1、aaCDR2和aaCDR3，其氨基酸序列分别为SEQ IDNO:4、SEQ ID NO:5、SEQ ID NO:6。
2.如权利要求1所述的抗HIgG-Fc单克隆抗体，其特征在于：所述抗体具有SEQ ID NO：7所示的重链区可变区氨基酸序列和SEQ ID NO:8所示的轻链区可变区氨基酸序列。
3.一种核酸分子，其特征在于：所述核酸分子包括编码如权利要求1-2任一所述的抗HIgG-Fc单克隆抗体。
4.一种细胞系，其特征在于：能够产生如权利要求1-2任一所述的抗HIgG-Fc单克隆抗体。
5.抗HIgG-Fc单克隆抗体的制备方法，其特征在于：所述方法包括，培养如权利要求4所述的细胞系，并产生所述的抗HIgG-Fc单克隆抗体。
6.抗HIgG-Fc单克隆抗体的应用，其特征在于：所述抗HIgG-Fc单克隆抗体在生物药品的开发中的应用。
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