新型冠状病毒核蛋白N端结构域的表达纯化以及晶体优化

Prokaryotic Expression,Purification and Crystallization of N-terminal Domain of Nucleocapsid Protein in SARS-CoV-2

本研究通过原核表达方法,探索新型冠状病毒核蛋白N端结构域的表达纯化,同时优化其蛋白晶体的生长条件,为新型冠状病毒核蛋白的结构生物研究提供基础。本研究合成N蛋白N端结构域基因序列,构建原核系统表达载体,利用大肠杆菌表达重组目的蛋白。Ni亲和层析和凝胶过滤层析的方法对N蛋白N端结构域重组蛋白进行纯化,SDS-PAGE电泳鉴定重组蛋白的纯度。本研究成功构建原核重组载体pET28b-N-N,大肠杆菌在37℃下进行扩增生长,16℃下进行诱导表达,80%目的蛋白以可溶蛋白的形式表达。Ni亲和纯化和凝胶过滤层析后,Quantity One软件进行蛋白胶积分分析,获得纯化后的积分占比,获得目的蛋白的纯度高达90%以上,利用Hampton的蛋白晶体试剂盒进行蛋白质晶体筛选,获得质量较高的N蛋白N端结构域蛋白质晶体。凝胶迁移试验(EMSA)验证了N蛋白N端结构域具有结合特定RNA片段的作用,明确了N蛋白N端结构域稳定新型冠状病毒基因组的功能。为进一步解析N蛋白N端结构域的三维结构以及后续抗体开发提供研究基础。

The expression and purification of the N-terminal domain of SARS-CoV-2 nucleocapsid protein(N-protein)were explored through prokaryotic expression system, and its growth conditions were optimized, aiming to provide a basis for structural biological research of SARS-CoV-2 nucleocapsid protein. The N-terminal domain gene sequence of the N protein was synthesized, the prokaryotic expression vector was constructed, and the recombinant target protein was expressed by Escherichia coli. The expressed products were purified by Niaffinity chromatography and gel filtration chromatography. The purity of the recombinant protein was identified by SDS-PAGE electrophoresis. The prokaryotic recombinant vector pET28b-N-N was successfully constructed. E. coli was amplified and grew at 37℃ and induced to express at 16℃,and 80% of the target protein was expressed in the form of soluble protein. The purity of the target protein was >90% via protein glue integral analysis and obtaining the integral ratio by Quantity One software after Niaffinity chromatography and gel filtration chromatography.The protein crystal was screened by Hampton protein crystallizer kit, and a high quality crystal of the N-terminal of the N-protein was obtained.Electrophoretic Mobility Shift Assay(EMSA)was used to qualitatively validate the definite interaction between the N-terminal of the N-protein with the specific RNA fragments, and confirmed the function of the N-terminal domain of N-protein stabilizing the SARS-CoV-2 genome. The results provide basis for the 3D structure of the N-terminal of the N-protein and the subsequent antibody development.