| As an in vitro display technology,ribosome display is a powerful tool for rapid screening of functional and specific protein molecules from large libraries.The broad application fields of this technology include antibody engineering,proteomics,dia g-nostics,and therapeutics.Rapid isolation and the directed evolution of high-affinity functional proteins,particularly antibodies,are the most powerful applications based on ribosome display.In this study,peripheral blood from two Xinjiang bactrian camels was used as the material to construct a ribosome-displayed natural nanobody library with2.38×10~144 individuals.Using pET-30a as an expression vector,a biologically active HIF-1?-PAS-B recombinant protein was successfully obtained through the renatura-tion of inclusion bodies.Using the HIF-1?-PAS-B recombinant protein as antigen,VHH-67 and VHH-212 with higher affinity were obtained after four rounds of selec-tion.Then,VHH-67 and VHH-212 were expressed in the periplasmic space and cyto-plasm of E.coli,respectively.Thermal stability analysis showed that the Tm value of VHH-67 was 76.60°C,and the Tm value of VHH-212 was 79.55°C.Fluorescence in-verted microscope observations and Western Blot results indicated that VHH-212 was expressed in pancreatic ductal adenocarcinoma cell lines.RT-qPCR results showed that VHH-212 bound HIF-1?protein intracellularly,which inhibited the function of HIF-1?transcriptional activation of downstream target genes to some extent.MTT colorimetric assays demonstrated that nanobodies had no toxicity on cells.In order to obtain nanobodies specifically against HIF-1?,a large ribo-some-displayed natural nanobody library was constructed.HIF-1?-PAS-B with screening quality was prepared as an antigen,and the selected nanobodies were sub-jected to activity evaluation.We hope that this study will contribute to the develop-ment of new drugs and diagnosis and treatment of diseases. |
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