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  • 秦照玲,彭浩然,丁翠玲,肖爱军,戚中田,赵平.严重急性呼吸综合征冠状病毒2核衣壳蛋白原核表达、纯化及其抗血清制备[J].第二军医大学学报,2020,41(5):481-486    [点击复制]
  • QIN Zhao-ling,PENG Hao-ran,DING Cui-ling,XIAO Ai-jun,QI Zhong-tian,ZHAO Ping.Prokaryotic expression, purification and antiserum preparation of severe acute respiratory syndrome coronavirus 2 nucleocapsid protein[J].Acad J Sec Mil Med Univ,2020,41(5):481-486   [点击复制]
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严重急性呼吸综合征冠状病毒2核衣壳蛋白原核表达、纯化及其抗血清制备
秦照玲1,彭浩然1,丁翠玲1,肖爱军2,戚中田1,赵平1*
0
(1. 海军军医大学(第二军医大学)海军医学系生物医学防护教研室, 上海 200433;
2. 上海捷瑞生物工程有限公司, 上海 201615
*通信作者)
摘要:
目的 表达并纯化重组的严重急性呼吸综合征冠状病毒2(SARS-CoV-2)核衣壳(N)蛋白,通过免疫小鼠制备SARS-CoV-2 N蛋白抗血清。方法 将含有SARS-CoV-2 N基因的pET28a-N原核表达质粒转化大肠杆菌BL21(DE3),用异丙基-β-D-硫代半乳糖苷诱导蛋白表达,用Ni-NTA亲和层析柱纯化SARS-CoV-2 N重组蛋白;将SARS-CoV-2 N重组蛋白联合锰佐剂通过肌内和皮下多点注射免疫BALB/c小鼠,获得抗血清;用蛋白质印迹分析检测SARS-CoV-2 N重组蛋白与SARS-CoV-2 N单克隆抗体及严重急性呼吸综合征冠状病毒(SARS-CoV)N多克隆抗体的反应;用间接免疫荧光实验检测小鼠抗血清与真核表达质粒转染细胞中SARS-CoV-2 N重组蛋白的反应。结果 成功诱导大肠杆菌表达出可溶性的SARS-CoV-2 N重组蛋白,相对分子质量约为55 000,纯化出该重组蛋白;蛋白质印迹分析结果显示SARS-CoV-2 N重组蛋白能与SARS-CoV-2 N单克隆抗体及SARS-CoV N多克隆抗体结合;间接免疫荧光实验检测结果表明制备的小鼠抗血清能与哺乳动物细胞中表达的SARS-CoV-2 N重组蛋白结合。结论 成功表达、纯化出SARS-CoV-2 N重组蛋白,并制备出该蛋白的小鼠抗血清,为后续建立SARS-CoV-2的快速诊断方法及开展SARS-CoV-2 N蛋白的功能研究奠定了基础。
关键词:  严重急性呼吸综合征冠状病毒2  核衣壳蛋白  原核表达  免疫血清
DOI:10.16781/j.0258-879x.2020.05.0481
投稿时间:2020-03-25修订日期:2020-04-07
基金项目:国家重点研发计划(2016YFC1200401),国家重大科技专项(2017ZX10304403-003).
Prokaryotic expression, purification and antiserum preparation of severe acute respiratory syndrome coronavirus 2 nucleocapsid protein
QIN Zhao-ling1,PENG Hao-ran1,DING Cui-ling1,XIAO Ai-jun2,QI Zhong-tian1,ZHAO Ping1*
(1. Department of Biomedical Defense, Faculty of Naval Medicine, Naval Medical University(Second Military Medical University), Shanghai 200433, China;
2. Shanghai Generay Biotech Co., Ltd, Shanghai 201615, China
*Corresponding author)
Abstract:
Objective To express and purify the recombinant nucleocapsid (N) protein of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), and prepare antiserum from immunized mice. Methods The prokaryotic plasmid pET28a-N containing SARS-CoV-2 N gene was transformed into Escherichia coli BL21 (DE3). The expression of recombinant SARS-CoV-2 N protein was induced by isopropyl-β-D-thiogalactopyranoside. The Ni-NTA affinity chromatography column was used to purify the recombinant SARS-CoV-2 N protein, and antiserum was obtained from the BALB/c mice immunized with recombinant SARS-CoV-2 N protein combined with manganese adjuvant through intramuscular and subcutaneous injections. The reactions of recombinant SARS-CoV-2 N protein with SARS-CoV-2 N monoclonal antibodies and severe acute respiratory syndrome coronavirus (SARS-CoV) N polyclonal antibodies were detected by Western blotting. The reaction of mouse antiserum with the recombinant SARS-CoV-2 N protein expressed in the cells transfected with eukaryotic expression plasmid was examined by indirect immunofluorescence assay. Results The recombinant SARS-CoV-2 N protein was successfully induced and expressed as a soluble protein with a molecular weight of about 55 000. High concentration of purified protein was obtained. The results of Western blotting showed that the recombinant SARS-CoV-2 N protein could be specifically recognized by the SARS-CoV-2 N monoclonal antibodies and the SARS-CoV N polyclonal antibodies. The prepared mouse antiserum could also correctly recognize the recombinant SARS-CoV-2 N protein expressed in mammalian cells by indirect immunofluorescence assay. Conclusion Recombinant SARS-CoV-2 N protein has been successfully expressed and purified from the prokaryotic expression system, and mouse antiserum has been prepared, which lays a foundation for establishing a rapid SARS-CoV-2 diagnostic tool and further studying the function of SARS-CoV-2 N protein.
Key words:  severe acute respiratory syndrome coronavirus 2  nucleocapsid protein  prokaryotic expression  immune sera