Preparation of anti-hCG single domain antibody by antibody grafting technique using an antigen-binding peptide.

Jing PENG; Qiong WANG; Xiaoling CHENG; Mengwen LIU; Mei WANG; Huawei XIN

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Preparation of anti-hCG single domain antibody by antibody grafting technique using an antigen-binding peptide.

Author: Jing PENG ¹ ; Qiong WANG ¹ ; Xiaoling CHENG ¹ ; Mengwen LIU ¹ ; Mei WANG ¹ ; Huawei XIN ¹
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1. College of Life Science and Health, Wuhan University of Science and Technology, Wuhan 430065, Hubei, China.
Publication Type:Journal Article
Keywords: CDR1; CDR3; expression and purification; fusion protein; grafted antibody; hCG; single domain antibody
From: Chinese Journal of Biotechnology 2018;34(4):569-577
CountryChina
Language:Chinese
Abstract: We used the antibody grafting technology to prepare anti-hCG single-domain antibodies on the basis of antigen-binding peptide to simplify the single-domain antibody preparation process and improving the biochemical stability of peptide. By using a universal single-domain antibody backbone (cAbBCII10), CDR1 or CDR3 was replaced by the hCG-binding peptide, and the grafted antibody gene sequences were synthesized and cloned into the prokaryotic expression vector pET30a(+) in fusion with a C-terminal sfGFP gene, i.e. pET30a-(His6)-cAbBCII10-CDR1/hCGBP1-sfGFP and pET30a-(His6)-cAbBCII10-CDR3/hCGBP3-sfGFP. The recombinant plasmids were transformed into E. coli BL21(DE3), and the fusion proteins were induced by IPTG. Highly soluble recombinant fusion proteins were obtained and purified by Ni-NTA affinity column. SDS-PAGE confirmed the purified protein as the target protein. The antigen-antibody binding assay showed that both the CDR1 and CDR3 grafted antibodies have hCG-binding activities. While the titers of the two grafted antibodies were similar, the binding affinity of CDR3 grafted antibody was higher than that of CDR1 grafted protein (about 2-3 times). The grafted antibodies retained the relatively high biochemical stability of the single-domain antibody backbone and were relatively thermostable and alkaline tolerant. The obtained antibodies also had a relatively high antigen-binding specificity to hCG. This study provided a reliable experimental basis for further optimization of anti-hCG single domain antibody by antibody grafting technology using antigen-binding peptide.