Research On Screening Peptide Mimics Of Ligands For Biomacromolecules Based On Phage Library

In vivo,most proteins perform their biological functions by Protein-Protein Interactions (PPIs),thus PPIs play indispensable roles in various areas.However,the identified PPIs are limited at present,and the development of researches puts forward a lot of demands on the discovery of new PPIs.Present research methods for PPIs have advantages,and also limitations.The latter results in that many research difficulties cannot be solved due to failure in discovery of PPIs,thus development of relative researches and applications are hindered,such as identification of targets of functional antibodies whose targets are unknown,immune tolerance of epitope-based vaccines,selection of biomarkers for the diseases difficult to diagnosis and selection of peptide binders with high affinity for biomacromolecules.To discover new PPIs,phage library-based selection of peptides to mimic ligands of biomacromolecules is a good strategy.Phage display library screening technology has been widely used in research on PPIs,however,above difficulties haven’t been solved with the following reasons.Owing to non-specific adsorption of phages to irrelevant materials,the percentage of target un-related phages (TUPs) is high and this results in inefficient screening.However,strategies to lower non-specific adsorption,proposed by some researches,still have limitations.Moreover,sanger sequencing is used for analyzing screened information in most studies,resulting in omission of many interactions.To improve the screened information,screened libraries were analyzed by next-generation sequencing (NGS) in few researches.However,NGS data analysis is tedious due to large data.To solve the difficulties,firstly,this study established a platform for phage screening with low non-specific adsorption.We optimized phage screening methods with low non-specific adsorption,including:(1) a rapid phage screening method based on solid-phase capture by ELISA plate,the method is rapid and specific,and has a superior advantage in depicting the serum antibody profiles;(2) alternate phage panning method with specificity and robustness,and this method lower the negative impacts from inhomogeneous distribution of library.The two methods are mutually complemented.We established a NGS data analysis platform for phage screening by writing MATLAB code,and realized the goal of "one-click" analysis from nucleic acid sequences of single samples to peptide distribution of total samples.We also established nucleic acid analysis system for phages.Based on phage screening platform with low non-specific adsorption,aiming at the above un-solved scientific problems,we concreted the detailed difficulties in our laboratory as entry points,explored and solved the difficulties and sore points,and figured out research strategies and laws.By using optimized phage screening technology,we firstly identified the target for a functional antibody whose target was unknown and failed to be identified.The platform identified that 4B12S mAb (a therapeutic antibody for pancreatic cancer)recognizes a linear epitope KDXLEXXR and its target is β chain of HLA-DR4 and HLA-DR7.This provided important foundations for studies on anti-tumor mechanism of 4B12S and HLA-DR as a new target in solid tumor treatment,also expanded the methods and provided important strategies for identifying targets for functional antibodies.Facing the problem of immune tolerance of epitope-based vaccine,we firstly used phage screening method to guide and simplify the design on immunogens for epitope focused immunization.We started from the unique epitope recognized by E6F6 and MID antibodies (therapeutic antibodies for Chronic Hepatitis B (CHB)).By screening binding sequences for the antibodies,this study further exactly clarified the core epitope.Then peptides were displayed on HBc183 VLP to be immunogens.BALB/c mice received sequential or mixed immunization produced high levels of antibodies against the therapeutic epitope.This provided a new strategy for researches from functional antibodies to vaccine design.On screening biomarkers for diseases which are difficult to diagnosis (such as tumors and autoimmune diseases),this study firstly combined phage display technology with immunosignature.Modeling on rheumatoid arthritis (RA) and nasopharyngeal carcinoma (NPC),we used peptides as antigen mimics to "discover"disease-related antigen-antibody interactions,and significantly distinguished the populations.This study laid the research foundation for new technology of screening biomarkers.On the study of screening ligand mimics with high affinity for macromolecular proteins,we modeled on SARS-CoV-2 Spike protein and firstly de novo selected a peptide binder (named Spep-1) with high affinity (KD=1.70～2.14 nM) and specificity for S2 subunit of Spike protein.Spep-1 based sandwich ELISA assay for detection of spike antigen in spiked human serum samples reached a lower limit of detection (LLOD)of femtomolar scale (760 fM) that increased the sensitivity more than 100 times comparing with similar studies.This study provided an effective measure for S2 subunit recognition,and also an important binder for future researches on virus entry,in-vivo virus tracing and targeted drug delivery,and provided a screening strategy for peptide binder with high-affinity.To sum up,this study successfully optimized two complementary phage screening methods with low non-specific adsorption,established "One-click" NGS data analysis platform,thus we greatly improved efficiency and success rate and also provided important technologies for other phage screening platforms.Based on the optimized platform,this study firstly solved several aspects of scientific problems which were assigned with detailed implications,prompted the development of related researches,explored research strategies and laws under multi-scenarios and will provide valuable research foundation and experience for future research.