Preparation Of Anti-hCG Single Domain Antibody By Antibody Grafting Technique Using An Antigen-binding Peptide

In this study we used the antibody grafting technology to prepare anti-hCG single-domain antibodies on the basis of antigen-binding peptide in purpose of simplifying the single-domain antibody preparation process and improving the biochemical stability of peptide.By using a universal single-domain antibody backbone(c AbBCII10),the 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 sf GFP gene,i.e.pET30a-(His6)-cAbBCII10-CDR1/hCGBP1-sf GFP and pET30a-(His6)-c AbBCII10-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.