Construction And Application Of A Noval Nanobody Fishing System Based On Bacterial-2-Hybrid

Nanobody(Nb),which is the variable domain of heavy chain of heavy-chain antibody,is the smallest antibody fragment with complete antigen binding ability.It has excellent properties such as strong stability,high solubility,strong tissue penetration,easy modification and humanization,which lay a solid foundation for its application in life sciences such as biosensors,disease diagnosis and treatment.Currently,conventional nanobody preparation relies mainly on phage display technology.However,this method is laborious and time-consuming as it requires purification of the antigen,as well as multiple enrichment to narrow the library to screen for affinity-sensitive nanobodies.To address these issures,here we established a novel nanobody screening system by using the“AND-gate”to screen for the colonies that habor the desired nanobodies,thus avoiding the low-immunization risk from toxic or low immunogenic antigens.Inspired by the“LexAWT/LexA408”dimeric repression system,the“AND-gate”was designed by fusing the target antigen(Bait)and nanobody(Prey)to the DNA binding domains of LexAWT and LexA408,respectively,so as to activate the resistance gene only if the Bait and Prey bind to each other with high efficiency.Basically,this nanobody-screening system contained two Escherichea coli compatible plasmids,the antigen-presenting plasmid pMHA2and the nanobody-displaying plasmid pMHN2,once the nanobody and antigen interact,they are prone to dimerize the antigen-LexAWT and nanobody-LexA408,thus forming the functional repressor for their target PsulA-cI857,thereby freeing the expression of P_R-kan~R as CI857 represses P_R promoter.As a proof of concept,we first applied the leucine zipper Fos-Jun to test the leackge of this system,which showed high stringency.Then the system was further verified using the sfGFP protein as antigen,NbsfGFP as the positive nanobody and cAbBCII10 as the negative control.The result whowed that only colonies harboring the sfGFP-NbsfGFP combination survived.Further more,a synthetic library with high VHH insertion rate and rich gene diversity was constructed by using trinucleotide cassettes,with a library capacity of 1.8×10~9 CFU/mL.Based on the screening system constructed above,a total of six specific antibodies with strong affinity were screened out for glycosylated hemoglobin HbA1c,tumor necrosis factor TNF-?and anthrax toxin protective antigen PA63,respectively.The affinities between these nanobodies and their respective antigens were further determined by ELISA(Enzyme-linked Immunosorbent Assay).The nanobody screening system and the protocals here described opened new strategy for the screening of the nanobodies,which not only simplified the nanobody generation process,but also reduced the preparation cost.