Preparation Of Anti-hCG Antibody-Like Molecule By Using A RAD Peptide Display System

Objective: Human chorionic gonadotropin is an important marker of pregnancy,pregnancy-related diseases and various cancers.It has important medical value for the research of hCG molecular immunoassay technology.On the basis of bioinformatics analysis,an anti-hCG antibody-like molecule was prepared by RAD peptide display system,and the antigen-binding activity of the grafted antibody-like molecule was tested to verify its application potential,which provided experimental support for further optimization of hCG clinical immunoassay technology.Methods: Bioinformatics tools were used to predict the structure and properties of RAD-grafted antibody protein.After linking to sfGFP gene,a gene of hCG-binding peptide-grafted RAD was synthesized and cloned into the prokaryotic expression vector pET30a(+)with His tag,and the bacterial expression plasmid(pET30a-RAD/hCGBP-sfGFP)was constructed.The recombinant plasmid was transformed into Escherichia coli BL21(DE3).The protein induced by IPTG was then purified by Ni-NTA affinity column,and identified by SDS-PAGE.After purification of the antibody-like protein,its binding affinity to hCG was determined by using a process of immunoabsorption followed by GFP fluorescence measurement.The comparisons of binding specificity and biochemical stability level of RAD-grafted antibody protein with a similarly grafted single-domain antibody based on a universal scaffold and commercial monoclonal antibody were performed.Result: Through bioinformatics analysis,the molecular weight of grafted RAD molecule was 58.93 kDa and its isoelectric point was 6.23.Besides,the molecule was hydrophilic proteins with good stability but did not have transmembrane regions and signal peptide sequences.By modeling of secondary and tertiary structure,it showed that the protein had a good spatial structure.The grafted RAD molecule with high purity was successfully prepared by prokaryotic expression experiments.The antibody-like protein which expressed well in E.coli was highly soluble,and the rapid production and purification were achieved.Biochemical experiments showed that RAD-grafted molecule has excellent hCG antigen-binding activity,while compared with other grafted protein and commercial monoclonal antibody prepared by classical pathways.Moreover,RAD-grafted molecule has relatively high binding specificity for hCG,but has some cross-binding activity with luteinizing hormone(LH).And it showed good biochemical stability in different temperature and pH environments.Conclusion: The anti-hCG antibody molecule was successfully prepared by RAD peptide display system,which has a good feasibility.Compared with commercial monoclonal antibody,RAD-grafted molecule has better antigen-binding activity and specificity for hCG molecules.And that molecule are significantly superior to single-domain grafted antibody,making it a good substitute for antibody with potential application.