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

doi:10.13560/j.cnki.biotech.bull.1985.2022-0400

摘要/Abstract

摘要：

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。Ni²⁺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。Ni²⁺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。

关键词: SARS-CoV-2, N蛋白, 纯化, 晶体

Abstract:

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 Ni²⁺ affinity 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 Ni²⁺ affinity 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.

Key words: SARS-CoV-2, nucleocapsid protein, purification, crystallization

陈铎, 刘永哲. 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[J]. 生物技术通报, 2022, 38(12): 149-155.

CHEN Duo, LIU Yong-zhe. Prokaryotic Expression，Purification and Crystallization of N-terminal Domain of Nucleocapsid Protein in SARS-CoV-2[J]. Biotechnology Bulletin, 2022, 38(12): 149-155.

图/表 6

表1 引物序列

Table 1 Primer sequences

引物名称 Primer name	引物序列 Primer sequence（5'-3'）
N-N-F	GGAATTCCATATGAATAATACTGCGTCTTGGTT
N-N-R	CCGCTCGAGTTACCCTTCTGCGTAGAAGCCTT

图1 SARS-CoV-2、SARS-CoV和MERS-CoV的N蛋白氨基酸序列对比

Fig. 1 Comparison of N protein amino acid sequences of SARS-CoV-2，SARS-CoV and MERS-CoV

图2 N-N蛋白基因片段扩增结果 1：PCR产物，M：DNA分子标准质量

Fig. 2 PCR amplification result of N-N protein 1：PCR amplification product. M：DNA molecular weight standard

图3 N-N蛋白的表达纯化 A：N-N蛋白通过Superdex 75凝胶色谱分子筛纯化图；B：纯化后的N-N蛋白SDS-PAGE结果图。M为蛋白质marker；1-3：N-N蛋白；4：诱导前样品；5：诱导后样品

Fig. 3 Expression and purification of N-N protein A：The N-N protein samples were purified by Superdex 75 column. B：The purified N-N protein samples were analyzed on SDS-PAGE. M：Protein marker. 1-3：The N-N protein；4：Before-induced.5：After-induced

图4 N-N蛋白与RNA结合的EMSA结果 1：空白RNA对照；2-7：N-N蛋白与RNA孵育（N-N蛋白浓度分别对应1、2、3、4、5、6 mg/mL）

Fig. 4 EMSA result of binding of N-N protein and RNA 1：Normal RNA control. 2-7：Incubation of RNA and N-N protein（The N-N protein concentrations correspond to 1，2，3，4，5 and 6 mg/mL respectively）

图5 N蛋白的晶体优化 A：PEG/Ion Screen 23号初筛晶体；B：添加Crystal Screen 45号复筛晶体；C：添加Additive Screen 25号最终获得晶体

Fig. 5 Optimization of N protein crystal A：Making Initial crystal by PEG/Ion Screen 23. B：Making further crystal by adding Crystal Screen 45. C：Making final crystal by adding Additive Screen 25

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