Construction And Evaluation Of A Fully Synthetic Scfv Library

Background:Phage display is laboratory techniques that allows scientists to study protein in-vitro interaction with other macromolecules on a large scale and select colnes with the hignest affinity of specific targets by an in vitro selection process called panning.By inserting the gene of target proteins into that of phage coat protein,the target proteins can be displayed on the sufface of bacteriophage after assembly,which resulting in a connection between genotype and phenotype.Phage display provides a means to identify target-binding proteins from a library of millions of different proteins without the need to screen each individual and then ampilify the selected clones in a process called in vitro selection,which is analogous to natural selection.As monoclonal antibodies have become the most important class of therapeutic biologicals on the market,"fully" human antibodies,screened from phage display library,were favored for its potentially superior clinical efficacy and lowest immunogenicity.Severe acute respiratory syndrome coronavirus 2(SARS-CoV-2)had caused over 4.8 million infectors by May 20th,2020.Using the library to screen the specific high-affinity antibodies can be a remarkable method for the invention of both diagnostic and therapeutic antibodies.Objective:In this study,we use light-chain DPκ9 and heavy-chain DP47 as frame to construct Single-chain variable fragment(scFv)phage display library.Then we screen for specific scFv that bind Procalcitonin(PCT)with high affinity to verify library capability of being using for development of therapeutic antibodies.Finally,using the library to screen for specific anti-SARS-CoV-2 antibodies.Methods:Synthesize degenerate primers of CDR3 and the gene of DPκ9-DP47-PⅢ Cd for construsting phagemid HP947S;Follow the instructions of Kunkel Mutagenesis to construct CCC-dsDNA;Prepare the electrocompetent TG1(M13K07)cells and electroporate CCC-dsDNA into it to construct the library then validate the capacity;Panning for specific anti-PCT scFv for 4 rounds of"binding-elution-amplification" in macroplates;Sequence the selected clones and analyse the amino acid sequence of CDR3 after Phage ELISA;Select the clones with hignest affinity to continue affinity maturation by firstly redefine CDRL1、CDRL2 and then CDRH1、CDRH2;Construct eukaryotic expression plasmid A2U-PsecX-scFv-hIgG1-Fc to express scFv-Fc then verify.Construct the expression vector of N protein and S protein of SARS-COV-2,purify some amounts of N protein and S protein for screening.Results:We successfully construct several scFv phage display libraries containing over one billion human antibody clones;obtain specific anti-PCT clones with high affinity(Kd≈50nM);Enhance the binding strength to 10nM after affinity maturation successfully construct eukaryotic expression plasmid A2U-PsecX,and express scFv-Fc in HEK293T cells.N protein was successfully expressed in E.coli.Conclusion:Our phage display libraries in this study are functional for panning for specific antibodies because we successfully obtain specific anti-PCT clones with high affinity(Kd≈6nM)after affinity maturation.We successfully construct eukaryotic expression plasmid A2U-PsecX-scFv-hIgG1-Fc to express scFv-Fc,which is fundamental for the purification of antibodies.Our work lays down a solid foundation for future screening for therapeutic antibodies.The expression of N protein and S protein of SARS-COV-2 brings great opportunity for the detection and therapy for COVID-19.