Selection And Identification Of Shark-derived Single-domain Antibodies Against Green Fluorescent Protein

Green fluorescent protein(GFP)is a kind of bioluminescent protein existing in marine coelenterates.It is widely used in life science researches and other fields due to its low toxicity and side effects,stable fluorescence signal,and easiness of detection.However,GFP suffers loss of fluorescence under some conditions,such as acidic environments,cryopreservation of tissue samples,and the use of histology reagents and fixatives.As an ideal tool,single-domain antibody(sdAb)can solve this problem.Shark-derived sdAb,also known as variable domain of immunoglobulin new antigen receptor(VNAR),is the smallest known antigen-binding unit.Its inherent advantages of low molecular weight,simple structure,high affinity,high stability and low autoimmunity have made it a research hotspot in the field.Therefore,shark-derived sdAbs against GFP will have wide application prospects and commercial values.In this thesis,the recombinant enhanced consensus green protein variant 123(eCGP123)was used as the antigen to immunizeChiloscyllium plagiosum.Peripheral blood was collected after third and fourth immunization.Two VNAR libraries with sizes of 3.4×10~8 CFU and 3.9×10~8 CFU were constructed.Positive phages were effectively enriched through three rounds of biopanning using phage display technology.A total of 36 VNAR sequences were screened and named as aGFP-1 to aGFP-36.Subsequently,the 36 VNAR sequences were constructed into pTT5-TEV-Fc eukaryotic expression vectors and transfected into HEK 293F cells.After 72 h of culture,the recombinant antibodies were purified by r Protein A affinity chromatography.The melting temperature(T_m)of the VNARs was determined by differential scanning fluorimetry,and the results showed that the T_m values of the 36 VNARs were ranging from 50℃to 65℃.The dissociation equilibrium constant(K_D)values were determined by biolayer interferometry technology,and the results showed that all VNARs had high affinity toward eCGP123,with K_Dvalues ranging from 6.76 nM to 605 nM.Among them,the affinity of aGFP-14 and aGFP-15reached the nanoscale level,with K_D values of 6.76 nM and 8.92 nM,respectively.Both VNARs competed with each other for binding with eCGP123 protein.Size exclusion chromatography and co-immunoprecipitation assay indicated that aGFP-14 and aGFP-15 formed stable complexes with eCGP123.In order to clarify the interaction between aGFP-14 and eCGP123,the crystal structure of eCGP123-aGFP-14 complex was analyzed by X-ray crystallography.It showed that the interaction force between eCGP123 and aGFP-14 was mainly the hydrogen bond formed by the amino acids in the binding interface.Enhanced green fluorescent protein(EGFP),another GFP variant,is usually used as a fluorescence tracker in microscopy imaging.Immunostaining assay revealed that aGFP-14 could specifically recognize EGFP expressed on the membrane of Hela cells and HEK 293T cells,as well as in mouse brain tissues.This study provides a theoretical reference for the application of GFP-specific sdAbs in microscopy imaging,subcellular localization,degradation of GFP fusion proteins and disease diagnosis.