生物技术通报 ›› 2022, Vol. 38 ›› Issue (12): 149-155.doi: 10.13560/j.cnki.biotech.bull.1985.2022-0400
收稿日期:
2022-05-23
出版日期:
2022-12-26
发布日期:
2022-12-29
作者简介:
陈铎,男,博士,研究方向:呼吸与危重症医学;E-mail: 基金资助:
Received:
2022-05-23
Published:
2022-12-26
Online:
2022-12-29
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。
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.
引物名称 Primer name | 引物序列 Primer sequence(5'-3') |
---|---|
N-N-F | GGAATTCCATATGAATAATACTGCGTCTTGGTT |
N-N-R | CCGCTCGAGTTACCCTTCTGCGTAGAAGCCTT |
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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