The Construction,Validation And Application Of A Synthetic Nanobody Library Based On Alpaca Natural Nanobody Guidance

Nanobody（Nb）,or variable domain of heavy chain antibody（VHH）,is a single-domain protein derived from the variable region of heavy chain antibodies,which is naturally produced by Camelidae.High tissue penetration,low immunogenicity,high water solubility,and stability are advantages that paved the way for Nb as an alternative affinity reagent to traditional antibodies.Nb is widely used in food safety testing,medical clinical diagnosis,and antibody biologics development.The conventional method of preparing Nbs is through animal immunization and library screening.Animal immunization is expensive,time-consuming,limited in screening,and not suitable to immunize strong toxicity or pathogenicity antigens.Native and synthetic libraries are more diverse and can be used for the screening of different antigens,providing strong support for the low-cost and rapid production of Nbs.In this study,an alpaca-derived（Vicugna pacos）native Nb phage display library,namely N-library,was constructed from PBMC collected from 13 healthy animals;a synthetic Nb library,namely S-library,was constructed under the guidance of the next-generation sequence data of natural Nbs.The two libraries were screened against five protein targets,including anti-aflatoxin B₁（G8）,the fusion protein of G8 and green fluorescent protein（G8-GFP）,bovine serum albumin（BSA）,ovalbumin（OVA）,and acetylcholinesterase（Ach E）,respectively.The main contents and findings were as follows:（1）Construction,identification,and screening validation of a native Nb library.A native phage display Nb library originating from 13 healthy Alpacas was constructed comprising 8.4×10⁷ independent transformants.Colony PCR of 24randomly picked clones shows that the VHH fragment insertion rate was 96%.Sanger sequencing shows 87%of 30 random clones possess intact Nb coding sequences,and no duplication was found.After being rescued by the helper phage KM13,the titer of the phage display library,N-library,reached 2.4×10¹² PFU/m L.Two recombinant proteins,Nb G8 and G8-GFP,were prokaryotic expressed and purified via affinity chromatography.An indirect competitive enzyme-linked immunosorbent assay（IC-ELISA）was conducted to evaluate the specificity for the Nb G8 and G8-GFP,with IC₅₀ 4.53 ng/m L and 6.42 ng/m L,respectively.Five protein targets（Nb G8,G8-GFP,BSA,OVA,and Ach E）were screened against the N-library using solid phase biopanning.Nb binders for each antigen were isolated,including 7anti-BSA,2 anti-OVA,4 anti-Ach E,and 1 anti-G8 framework region nanobody.（2）Design,construction,and screening validation of a sequence-based synthetic Nb library.Next-generation sequencing（NGS）analysis based on alpaca-derived natural nanobody sequences to obtain the frame sequence IGHV3S65*01-IGHJ4*01 of a synthetic nanobody library.Sequence analysis of the germline genes using IGHV3S65*01-IGHJ4*01 showed that the average length of the complementarity determining region 3（CDR3）was 19 amino acids.The distribution of amino acids at each position of CDR3 were counted.Diversity was introduced in the CDR3 region by designing condensed primers,and the complete nanobody coding region was obtained by one-step overlapping extension PCR to construct a synthetic library containing2.19×10⁸ independent transformants,named S-library.After being rescued by the helper phage M13K07,the titer of the phage display library reached 1.5×10¹⁴ PFU/m L.Colony PCR of 46 randomly picked clones shows that the VHH fragment insertion rate was 96%.Sanger sequencing shows 96%of 50 random clones possess intact Nb coding sequences,and no duplication was found.A further NGS analysis was performed on12,102,052 sequences,92.46%of which contained a CDR3 region of 19 amino acids in length,as expected by design,and a sequence diversity of 87.45%.Five protein targets（Nb G8,G8-GFP,BSA,OVA,and Ach E）were screened against the S-library using solid phase biopanning.Nb binders for each antigen were isolated,including 5anti-BSA,4 anti-OVA,3 anti-Ach E,and obtained a highly enriched strain of sequence G1 encoding a binding Nb G8 nanobody.（3）Expression,purification,and bioactivity identification of Nbs.Seven representative nanobody monomers from two libraries were expressed in the E.coli expression system（Four varieties of N-library sources,three varieties of S-library sources）.The SDS-PAGE results showed that both obtained about 20 KDa of recombinant protein.Antigen-binding activity was identified by indirect ELISA,and Nb E22anti-Nb G8,B16anti-BSA,N2anti-Ach E,N18anti-Ach E from N-library and Nb G1anti-Nb G8from S-library all specifically bound to the corresponding antigens.The checkerboard titration test for Nb B9_anti-BSA and S4_{anti-Ach E} from S-library showed lower color development,indicating poor binding.The affinity of four nanobodies was determined by bio-layer interferometry（BLI）,three of which were from N-library,Nb E22_{anti-Nb G8},N2_{anti-Ach E} and N18_{anti-Ach E},and one from S-library,Nb G1_{anti-Nb G8},with an affinity range of 300 n M to 30 n M.The bioactive nanobodies were obtained in both libraries,especially in the synthetic library,and it is expected that further design and modification of high-affinity nanobodies and construction of synthetic libraries with larger library capacity and diversity will lead to the screening of more nanobody candidates against different target antigens.