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

doi:10.13523/j.cb.2110009

China Biotechnology

2022, Vol. 42

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

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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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

Received: 09 October 2021 Published: 05 May 2022

ZTFLH:

Q816

Corresponding Authors: Xiao-jun WANG,Ya-yu WANG E-mail: wangxiaojun@caas.cn;wangyayu1987@163.com

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	CHEN Yang
	LIU Tong
	ZHANG Jia-qi
	LIAO Hua-xin
	LIN Yue-zhi
	WANG Xiao-jun
	WANG Ya-yu

Cite this article:

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.

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

Fig.1 Schematic diagram of preparation of McAbs based on single B cell antibody technology

Fig.2 Construction strategy of immunogenic protein expression vector

Fig.3 Expression and identification of horse IgG1-C protein M: Marker;1, 3:EqIgG1-C nonreducing state;2, 4:EqIgG1-C reducing state; 1, 2 SDS-PAGE detection results;2, 4 Western blot detection results

Fig.4 Antibody titers test results of immunized mice

Fig.5 Sorting of EqIgG1-C protein specific memory B cells by flow cytometry

Fig.6 PCR amplification of heavy/light chain variable region (a) Heavy chain variable region (b)Kappa chain variable region

Fig.7 Expression and identification of EqIgG1-C specific mAb (a) Detection of 3 recombinant antibodies 1-3: IgG561, IgG563, IgG641(non-reduced); 4-6: IgG561, IgG563, IgG641(reduced) (b)Westen blot detection of 3 antibodies 1, 3, 5: EqIgG1-C reduced; 2, 4, 6: EqIgG1-C non-reduced; 1,2: Detected by IgG561; 3,4: Detected by IgG563; 5,6: Detected by IgG641 (c)Detection of different recombinant antibody titers


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