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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-jun2,***( ),WANG Ya-yu1,***( ) |
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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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 Expi293FTM 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.
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Received: 09 October 2021
Published: 05 May 2022
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Corresponding Authors:
Xiao-jun WANG,Ya-yu WANG
E-mail: wangxiaojun@caas.cn;wangyayu1987@163.com
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