Objective To obtain RSPO 1-binding nanobodies from the peripheral lymphatic blood of immunized camels by phage display technology,construct RSPO1 phage display library after purification,and perform immunopanning.Methods RSPO1 gene was connected with pCMV-Fc vector to construct the plasmid RSPO1-pCMV-Fc,which was transiently trans-fected into HEK-293F cells to express RSPO1 protein.RSPO1 protein was purified by Protein A gel column,HiLoadTM 16/600 SuperdexTM 200pg column and SuperdexTM 24 Increase10/300 GL column in turn,then used to immunize camels,and the peripheral blood was collected for isolating the lymphocytes.The cellular RNA was extracted,and cDNA was synthe-sized by reverse transcription.The VHH fragment was amplified by two-step nest PCR,and cloned into pMECS phage vector to construct phage display library.After two rounds of panning,the phages binding to RSPO1 were gathered,and then iden-tified by ELISA and sequenced.Results The plasmid RSPO 1-pCMV-Fc was constructed correctly as identified by PCR and sequencing.The relative molecular mass of expressed RSPO1 protein was about 172 000,with the purity of about 70％.The RSPO1 phage display library was 1.2 × 108 cfu,and the enrichment degree of phages reached 12 after two rounds of panning.A total of 19 nanobody sequences were obtained.Conclusion In this study,a good diversity of nanobody sequen-ces were obtained,which provides a possibility for understanding the Wnt signaling pathway related to RSPO 1.
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