Application Of Affinity Maturation In Natural Protein And Synthetic Nanobody

Affinity maturation is a process in which the immune system produces antibodies with higher affinity in the process of responding to antigens.This process depends on the random mutation of antibody variable region and the screening of antibodies by in vivo cells.At present,in vitro affinity maturation technology is widely used in protein engineering,aiming at improving the interaction between molecules.We optimized the SH2 domain of phosphotyrosine kinase by affinity maturation technology to make it have high affinity to bind to tyrosine sulfate.Through this technology,we optimized the nanobody C5 and obtained SARS-CoV-2 neutralizing antibody with high affinity.Sulfated modification of tyrosine is a common post-translational modification,which participates in a series of important biochemical reactions in vivo.The sulfation modification of tyrosine is related to the occurrence of some diseases,but the lack of tools to enrich tyrosine sulfation modification protein leads to the lack of research on sulfation modification protein.The native SH2 domain has the ability to bind phosphotyrosine,and the sulfated group is highly similar to the phosphorylated group.Based on this feature,the SH2 domain was modified and mutated to obtain some SH2 variants that bind sulfatyrosine.In addition,we also improved the conventional screening stratagem which obtained positive clones by random selection.Because of the limitation of the conventional screening stratagem,some variants with high affinity can’t be obtained.There are many reasons contributing to this result,for example,some variants with low base or slow growth rate.NGS has the advantages of large flux,high precision and abundant information.Using the advantages of NGS data can reduce the randomness of experiments.We combined conventional library screening with NGS data analysis to make up for the defects of conventional library screening.According to NGS data,the change of amino acid ratio and the growth trend of each variant in the screening process were analyzed,and the enrichment value of each variant was calculated according to the defined enrichment index "EI".After that,we predicted potential superbinder of sulfated tyrosine.Finally,we identified a sulfatyrosine superbinder SH2-4 by enzyme-linked immunosorbent assay and Bio-Layer Interferometry technique.COVID-19 broke out in 2019.This disease was caused by SARS-CoV-2.The spread of SARS-CoV-2 not only affects the development of human society,but also threatens human life and health.SARS-CoV-2 belongs to RNA virus,which enters the host cell by combining the Spike protein in the protein coat with the angiotensin converting enzyme 2(ACE2)on the surface of the host cell.This interaction provides an important breakthrough point for the design of vaccines and neutralizing antibodies.In this study,we obtained a nanobody,which can bind Spike protein in SARS-CoV-2 protein coat and block its binding with ACE2 on the surface of host cells,thus achieving preventive and therapeutic effects.Firstly,we used Spike,Receptor Binding Domain(RBD)of Spike and S1 domain of Spike to screen the phage display library of synthesized nanobody,and obtained a variety of nanobodies that bind to different epitopes of spike.Through epitope competition experiment,we found that nanobody C5 has neutralizing ability,but because of its poor affinity level and low protein yield,we optimized nanobody C5 by constructing affinity mature library,and successfully obtained C5D2 and C5G2 with high affinity,strong neutralizing ability and high protein yield.