The Design,Construction And Idetification Of A Fully Synthetic Nanobody Library

The synthetic antibody libraries were based on a set of optimized frameworks.Synthetic elements were introduced into the natural space of antibodies.It has advantages of large sequence diversities and large library size.The synthetic antibody library becomes a supplement of immune library or na?ve library and has a crucial fuction on the development of antibody drugs.In this study,a fully synthetic nanobody library was construced and identified.To verify the biological fuctions of the nanobody library,anti-HIF-1?-PAS-B nanobodies were screened.Firstly,this fully synthetic nanobody library was based on the framework originating from a well-expressed Na?ve nanobody library.The sequence of frame and the numbers of amino acid in the CDRs were determined through analyzing the sequence of Na?ve nanobody library.The sequence of VHH was synthesized and inserted into the phagemid vector pCantab 5E.Secondly,diversity was introduced in the CDRs through the use of the degenerate codon NNK.The VHH fragments were constructed by overlapping PCR,cloned into the phagemid vector pCantab 5E and electro-transformed into Escherichia coli TG1 cells.The synthetic nanobody library was achieved by scrapping colones from the plates.The total size of the library was4.2×10¹⁰ colony-forming units?CFU?.The sequencing results of the 100 randomly picked clones showed the library had a high diversity and a complete VHH insert of82%.Finally,anti-HIF-1?-PAS-B nanobodies were screened from the synthetic nanobody library after four rounds panning by phage display technology to identify the fully synthetic nanobody library.