Construction Of Anti-Cry1Ab Peptide And Its Application In Electrochemical Immunosensor

With the widespread application and rapid development of immunoassay technology in food safety,clinical diagnosis,and detection of environmental contaminant residues,higher requirements are placed on the preparation of immunological recognition element that are critical in immunoassays,and all kinds of recognition elements emerge as the times require.Peptide molecules have become one of the hotspots in recent research with their many advantages,and they are expected to become a potential new recognition elements.The low molecular weight of the peptide makes it particularly accurate for the recognition of target molecules and can reached into the hidden sites of the target protein that cannot be recognized by monoclonal antibodies.Compared to the preparation of traditional monoclonal antibodies with long cycle times,high difficulty and high costs,the preparation of polypeptide molecules is rapid,easy to modify,and inexpensive.In addition,immunoassay methods for large-molecule antigens（proteins,bacteria,viruses,etc.）are usually based on double-antibody sandwich methods.Obtaining paired antibodies against different epitopes is the premise and basis for establishing a double antibody sandwich immunoassay system.Due to small molecular weight peptides with the precise and diverse recognition sites,they can be randomly paired with the monoclonal antibodies,thus avoiding difficulty of antibody matching in sandwich immunoassay.In this study,using the transgenic protein Cry1Ab as a model,a peptide molecular recognition element that specifically recognizes Cry1Ab was prepared,based on the technology of phage display peptide library for panning and recombinant plasmid construction and expression.And it is applied to the electrochemical sensing platform to realize the ultrasensitive immunoassay for Cry1Ab.It provides a new idea for the preparation of immune recognition elements and the establishment of immunoassay methods for other types of macromolecular antigens.The main research results are as follows:1.Four phage displayed peptides（Ab-1,Ab-18,Ab-39,Ab-48）that specifically recognize the Cry1Ab protein were obtained using phage panning technology.The sandwich Phage-ELISA standard curves based on Ab-1,Ab-18,Ab-39 and Ab-48were established with SC₅₀ values of 398.42 ng/mL,127.81 ng/m L,138.89 ng/m L,148.52 ng/mL,respectively.2.An anti-Cry1Ab peptides gene consisting of three specific 12-peptide genes（Ab-39,Ab-48 and Ab-18）and linked by rigid linkers was designed and synthesized.Then it was connected to the Fc-terminal gene of the mouse IgE antibody by overlap extension PCR,and the recombinant expression vector“pET25b-Peptides-Fc”was constructed.The recombinant plasmid was transformed into E.coli Rosetta competent cells and induced by 0.1 mM IPTG to express the polypeptide chimeric antibody Peptides-Fc.3.The sandwich ELISA assay based on Peptides-Fc was constructed using the chimeric antibody Peptides-Fc as the capture antibody and the phage displayed peptides Ab-39 as the detection antibody.The sandwich Phage-ELISA standard curve was performed with the SC₅₀ value of 228.56±10.82 ng/mL and the linear range of97.29-536.97 ng/m L.4.Based on phage display peptide Ab-39,an electrochemical immunosensor was established to achieve ultrasensitive detection of Cry1Ab protein.The gold nanoparticles were used to modify the gold electrode,and the anti-Cry1Ab monoclonal antibody 8G2 was immobilized onto the electrode surface through a covalent bond.Then the phage display peptide Ab-39 was used as the detection antibody to form a sandwich layer of“mAb 8G2-Cry1Ab-Ab39”onto the electrode surface.The peak current corresponding to different concentrations of Cry1Ab protein was captured by square wave voltammetry detection mode,based on the relationship,a standard curve was established with a linear range of 0.01-100 ng/m L and a detection limit of 0.007 ng/mL.The recoveries of the spiked corn and wheat samples were 90%to 120%and 86.7%to 120%,respectively.