基于单个B细胞抗体基因扩增技术筛选马IgG1单克隆抗体<sup>*</sup>

doi:10.13523/j.cb.2110009

中国生物工程杂志

2022, Vol. 42

Issue (4): 17-23 DOI: 10.13523/j.cb.2110009

技术与方法

基于单个B细胞抗体基因扩增技术筛选马IgG1单克隆抗体^*

陈阳^1**,刘彤^1**,张佳琦²,廖化新¹,林跃智²,王晓钧^2,^***(

),王亚玉^1,^***(

)

1 暨南大学生命科学技术学院广州 5106321
2 中国农业科学院哈尔滨兽医研究所兽医生物技术国家重点实验室/马传染病和慢病毒病研究创新团队哈尔滨 150069

Screening of Monoclonal Antibodies Targeting the Equine IgG1 Based on Single B Cell Antibodies Gene Amplification Technology

CHEN Yang¹,LIU Tong¹,ZHANG Jia-qi²,LIAO Hua-xin¹,LIN Yue-zhi²,WANG Xiao-jun^2,^***(

),WANG Ya-yu^1,^***(

)

1 Department of Cell Biology & Institute of Biomedicine, College of Life Science and Technology, Jinan University, Guangzhou 5106321, China
2 State Key Laboratory of Veterinary Biotechnology, Harbin Veterinary Research Institute,the Chinese Academy of Agricultural Sciences, Harbin 150069, China

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摘要：

在马的免疫学研究领域中,由于目前市场上缺乏商业化的马IgG单克隆抗体,使得对马的B细胞研究受到很大阻碍,IgG是B细胞受体(BCR)的重要构成成分,与B细胞分化成熟相关。为了获得马IgG特异性单克隆抗体,利用单个B细胞扩增技术进行抗体筛选。首先,将马IgG蛋白(EqIgG1-C)密码子优化后合成到真核表达载体pcDNA3.4上,纯化出抗原蛋白。随后,使用蛋白质免疫小鼠,分离脾细胞后利用流式细胞术分离特异性单个B细胞,扩增出抗体重链和轻链的可变区基因,用overlapping PCR方法扩增出线性化的完整抗体,并进行鉴定。结果从80个B细胞中获得了27株特异性重组单克隆抗体,并挑选出3株线性结合活性最强的抗体基因构建到表达载体上,共转染Expi293F^TM细胞后表达纯化,经过ELISA和Western blot验证,显示获得的抗体可以和EqIgG1-C蛋白有良好的结合作用。使用该方法可以省时高效的获得特异性抗体,为马的免疫学研究提供了重要研究工具,为鼠单克隆抗体筛选提供了技术拓展。

关键词： 马IgG; 单个B细胞PCR; 抗体筛选技术; 单克隆抗体

Abstract:

In the field of equine immunology, research on equine B lymphocytes has been greatly hampered by the lack of commercial monoclonal antibodies of IgG. IgG is an important component of B cell receptor (BCR), which is associated with the differentiation and maturation of B cells. In order to obtain specific monoclonal antibodies of equine IgG, single B lymphocyte amplification was used to screen the antibodies. Firstly, the codon of equine IgG protein (EqIgG1-C) was optimized and synthesized on eukaryotic expression vector pcDNA3.4, and the antigenic protein was purified. Subsequently, the mice were immunized with the protein, and after the spleen cells were separated, the specific single B lymphocyte was separated by flow cytometry. The variable region genes of heavy and light chain of antibody were amplified by overlapping PCR method, and the complete antibody was identified. Finally, 27 strains of specific recombinant monoclonal antibodies were obtained from 80 B cells, and 3 strains with the strongest linear binding activity were selected and constructed into expression vector. After co-transfection of Expi293F^TM cells, antibodies were expressed and purified. Verified by ELISA and Western blot, the results showed that the antibodies has extraordinary binding activity to EqIgG1-C protein. Using this method can save time and obtain specific antibodies efficiently, which provides an important research tool for the study of equine immunology.

Key words: Equine IgG Single B cell PCR Antibody screening technology Monoclonal antibody

收稿日期: 2021-10-09 出版日期: 2022-05-05

ZTFLH:

Q816

基金资助: * 兽医生物技术国家重点实验室开放课题基金(SKLVBF2017)

通讯作者: 王晓钧,王亚玉 E-mail: wangxiaojun@caas.cn;wangyayu1987@163.com

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引用本文:

陈阳, 刘彤, 张佳琦, 廖化新, 林跃智, 王晓钧, 王亚玉. 基于单个B细胞抗体基因扩增技术筛选马IgG1单克隆抗体^*[J]. 中国生物工程杂志, 2022, 42(4): 17-23.

CHEN Yang, LIU Tong, ZHANG Jia-qi, LIAO Hua-xin, LIN Yue-zhi, WANG Xiao-jun, WANG Ya-yu. Screening of Monoclonal Antibodies Targeting the Equine IgG1 Based on Single B Cell Antibodies Gene Amplification Technology. China Biotechnology, 2022, 42(4): 17-23.

链接本文:

https://manu60.magtech.com.cn/biotech/CN/10.13523/j.cb.2110009 或 https://manu60.magtech.com.cn/biotech/CN/Y2022/V42/I4/17

图1 单个B细胞抗体筛选平台技术原理

图2 免疫原蛋白表达载体构建策略

图3 EqIgG1-C蛋白表达鉴定

图4 免疫小鼠的抗体表达水平检测

图5 流式细胞仪分选EqIgG1-C蛋白特异性记忆B细胞

图6 抗体可变区重链(a)/轻链(b)PCR扩增

图7 IgG1-C特异性单克隆抗体的表达与鉴定

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