SARS-CoV-2核衣壳蛋白预测与原核重组表达应用

Prediction and prokaryotic expression of SARS-CoV-2 nucleocapsid protein

为了优化构建SARS-CoV-2核衣壳蛋白原核表达载体并获得大肠杆菌表达的重组融合蛋白,研究利用公开的基因组数据与在线各蛋白质预测工具分析SARS-CoV-2核衣壳蛋白与其他β-CoV核衣壳蛋白的异同,以及二、三级结构情况;为合成、构建大肠杆菌表达系统密码子优化的SARS-CoV-2核衣壳蛋白原核表达载体提供辅助。基于生物信息分析,设计并构建含His标签的重组原核载体pET-N,转化BL21大肠杆菌感受态细胞,IPTG诱导表达目的蛋白,亲和层析法纯化重组融合蛋白,并经SDS-PAGE与Western Blot进行鉴定。预测分析了SARS-CoV-2核衣壳蛋白的序列特征与结构情况;并成功获得原核表达重组载体pET-N,发现其重组融合蛋白在上清液与包涵体中都存在,通过破碎包涵体,纯化获取目的蛋白,为间接ELISA法检测感染者血清中抗体所需的ELISA包被抗原提供参考。

In order to optimize the construction of prokaryotic expression vector of SARS-CoV-2 nucleocapsid protein and obtain the recombinant fusion protein expressed in Escherichia coli,we first analyzed the sequential similarities between SARS-CoV-2 nucleocapsid protein and otherβ-CoV nucleocapsid proteins,as well as the secondary and tertiary structures using public genomic data and protein prediction tools.Then,a recombinant vector pET-N with His tag was constructed and transformed into BL21 cells.The target protein was next induced and expressed by IPTG.Finally,the recombinant fusion protein was purified by affinity chromatography and identified by both SDS-PAGE and Western Blot.We found that the recombinant fusion protein existed in both the supernatant and the inclusion body,as well the target protein was purified by breaking the inclusion body.The recombinant fusion protein provides an alternative way of detecting the antibody in the serum of infected persons at the full advantages of the bioinformatics methodology.