Construction Of Nanobody Library And Screening Of Anti-Hypoxia Inducible Factor-1(HIF-1?) Nanobody By Ribosome Display

Ribosome display is a novel in vitro selection and evolution technology for proteins and peptides from large libraries. It utilizes formation of the mRNA-ribosome-polypeptide ternary complex in a cell-free protein synthesis system to link genotype(mRNA) to phenotype(polypeptide), and generates high-affinity binders from libraries of folded proteins. It has been successfully applied to single-chain Fv fragments of antibodies. Nanobodies( or VHHs) have been proven to be a considerable potential tool for experiment research and medical applications, attributed to their favorable properties such as small size, high solubility and solubility, together with low immunogenicity. This study describes the construction of a large naive VHHs library by ribosome display, and allows efficient screening binders for the PAS-B of hypoxia-inducible factor-1?..This work is a first step towards the feasibility of ribosome display nanobody library in our lab and lays the foundation for the discovery of new antibody drugs.In this protocol, total RNA was isolated from 4×107 leukocytes of freshly drawn,peripheral blood from alpaca(Australia). A total of 10.4 ug RNA was used for cDNA synthesis. VHH fragments were amplified by PCR and a spacer sequence(gene?, from the helper phage M13KO7), while introducing ribosome display elements, restriction sites, his-tag, etc. Then, VHH fragments and gene? were arranged randomly by SOE PCR(splicing by overlap extension PCR). Sequencing results showed that the library diversity and integrity is fine with a capacity of 4.9× 1012.Meanwhile, western blot analysis verified the translation possibility of the library. Expression vector pGEX-4T-1-HIF-1?-PAS-B in E.coli BL21 successfully expressed HIF-1?-PAS-B domain fusion with a GST-tag, following purified by GST-tag affinity chromatography, fusion protein GST-HIF-1?-PAS-B was digested by thrombin and purified again to achieve the antigen HIF-1?-PAS-B. After four cycles of panning, HIF-1?-PAS-B-specific VHHs were enriched and then cloned into Pcantab5 E for Phage Elisa. Finally, HIF-1?-PAS-B-specific VHHs were identified.