RSPO1蛋白的纯化及其免疫淘选

Purification and immunopanning of RSPO1 protein

目的通过噬菌体展示技术,从免疫骆驼的外周淋巴血液中获得与RSPO1结合的纳米抗体,纯化后构建RSPO1噬菌体展示文库,并进行免疫淘选。方法将RSPO1基因与pCMV-Fc载体连接后,构建质粒RSPO1-pCMVFc,瞬时转染HEK-293F细胞,表达RSPO1蛋白;经Protein A凝胶柱、HiLoad^(TM)16/600 Superdex^(TM)200pg柱、Superdex^(TM)24 Increase10/300 GL柱依次纯化RSPO1蛋白后,免疫骆驼,收集外周血并分离淋巴细胞;提取细胞RNA,反转录合成cDNA,通过2步巣式PCR扩增VHH片段,克隆至pMECS噬菌粒载体中,构建噬菌体展示文库,经2轮淘选使与RSPO1结合的噬菌体得到聚集后,进行ELISA鉴定并测序。结果质粒RSPO1-pCMV-Fc经PCR及测序鉴定证明构建正确。表达的RSPO1蛋白相对分子质量约172000,纯度约为70%;RSPO1噬菌体展示文库库容为1.2×10^(8)cfu,经2轮淘选使噬菌体富集度达到12;共获得19个纳米抗体序列。结论获得了多样性良好的纳米抗体序列,为了解与RSPO1相关的Wnt信号通路提供了可能。

Objective To obtain RSPO1-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 transfected into HEK-293F cells to express RSPO1 protein.RSPO1 protein was purified by Protein A gel column,HiLoad^(TM)16/600 SuperdexTM200pg column and Superdex^(TM)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 synthesized 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 identified by ELISA and sequenced.Results The plasmid RSPO1-pCMV-Fc was constructed correctly as identified by PCR and sequencing.The relative molecular mass of expressed RSPO1 protein was about 172000,with the purity of about 70%.The RSPO1 phage display library was 1.2×10~8cfu,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 sequences were obtained,which provides a possibility for understanding the Wnt signaling pathway related to RSPO1.