Toll样受体5激动剂CBLB502-Fc融合蛋白的表达纯化及初步鉴定

Expression, Purification and Preliminary Identification of Toll-Like Receptor 5 Agonist CBLB502-Fc Fusion Protein

目的:构建Toll样受体5激动剂CBLB502-Fc融合蛋白的真核表达载体,并在CHO细胞中表达,纯化得到具有生物学活性的目的蛋白。方法:首先从合成的pUC57-CBLB502质粒中扩增出CBLB502基因片段,酶切后克隆至pUC57-Fc质粒中,然后双酶切pc DNA3.1和pUC57-CBLB502-Fc,将CBLB502-Fc基因片段克隆至载体pc DNA3.1,挑选阳性克隆并测序,再将重组质粒转染CHO细胞,筛选高表达细胞系,用免疫印迹鉴定表达情况,Protein A亲和柱纯化目的蛋白,SDS-PAGE鉴定目的蛋白,用小鼠辐射试验初步验证目的蛋白的生物学活性。结果:构建了pc DNA3.1-CBLB502-Fc真核表达载体;转染CHO细胞,筛选得到CBLB502-Fc高表达细胞系,并经免疫印迹鉴定培养上清;纯化得到较高纯度的融合蛋白并经SDS-PAGE鉴定;小鼠辐射实验表明CBLB502-Fc具有抗辐射作用。结论:真核表达并纯化了具有抗辐射作用CBLB502-Fc融合蛋白,为后续研究CBLB502-Fc的生物学功能奠定了重要基础。

Objective：To obtain CBLB502-Fc fusion protein with biological activity by introducing a designed eukaryotic expression vector into CHO cells.Methods：The CBLB502 gene was amplified from pUC57-CBLB502 plasmid by PCR and cloned into pUC57-Fc vector.Then the CBLB502-Fc was inserted into the pcDNA3.1 vector after double enzyme digestion.The positive clones were picked up and subjected to sequencing.The recombinant plasmid pcDNA3.1-CBLB502-Fc was transfected into CHO cells.Then the highly expressed cell lines were screened.The target protein was purified by Protein A affinity column and identified by SDS-PAGE and Western blotting.The activity of fusion protein was detected by radiation test in mice.Results：The eukaryotic expression vector of pcDNA-CBLB502-Fc was successfully constructed.The cell lines with high expression of CBLB502-Fc fusion protein was screened.The fusion protein was purified by Protein A affinity column and confirmed by Western blotting.The radiation test of mice was made to determine the anti-radiation effect of CBLB502-Fc.Conclusion：We have purified the fusion protein CBLB502-Fc eukaryotic expressed,which layed the important foundation of further study on its biological function.