严重急性呼吸综合征冠状病毒2型核衣壳蛋白的原核表达、纯化及抗血清制备

Q4 Medicine

海军军医大学学报 Pub Date : 2020-01-01 DOI:10.16781/J.0258-879X.2020.05.0481

Qin Zhao-Ling, Peng Haoran, D. Cui-ling, Xiao Ai-Jun, QI Zhong-tian, Z. Ping

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引用次数: 1

摘要

目的表达和纯化重组SARS-CoV-2病毒核衣壳(N)蛋白，并从免疫小鼠制备抗血清。方法将含有SARS-CoV-2 N基因的原核质粒pET28a-N转化至大肠杆菌BL21 (DE3)，用异丙基-β- d -硫代半乙酰核苷诱导表达重组SARS-CoV-2 N蛋白，采用Ni-NTA亲和层析柱纯化重组SARS-CoV-2 N蛋白。通过肌肉和皮下注射重组SARS-CoV- 2n蛋白联合锰佐剂免疫BALB/c小鼠，获得重组SARS-CoV- 2n蛋白与SARS-CoV- 2n单克隆抗体和严重急性呼吸综合征冠状病毒(SARS-CoV) N多克隆抗体的反应，采用Western blotting检测小鼠抗血清与转染细胞中表达的重组SARS-CoV- 2n蛋白的反应结果成功诱导重组SARS-CoV- 2n蛋白，并表达为分子量约为55000的可溶性蛋白，获得了高浓度的纯化蛋白。Western blotting结果表明，重组SARS-CoV- 2n蛋白可被制备的小鼠SARS-CoV- 2n单克隆抗体和SARS-CoV- 2n多克隆抗体特异性识别结论在原核表达系统中成功表达并纯化了重组sars - cov - 2n蛋白，制备了小鼠抗血清，为建立快速诊断工具和进一步研究sars - cov - 2n蛋白的功能奠定了基础

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Prokaryotic expression, purification and antiserum preparation of severe acute respiratory syndrome coronavirus 2 nucleocapsid protein

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

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来源期刊

海军军医大学学报 Medicine-Medicine (all)

CiteScore

0.50

自引率

0.00%

发文量

14752

期刊介绍： Founded in 1980, Academic Journal of Second Military Medical University(AJSMMU) is sponsored by Second Military Medical University, a well-known medical university in China. AJSMMU is a peer-reviewed biomedical journal,published in Chinese with English abstracts.The journal aims to showcase outstanding research articles from all areas of biology and medicine,including basic medicine(such as biochemistry, microbiology, molecular biology, genetics, etc.),clinical medicine,public health and epidemiology, military medicine,pharmacology and Traditional Chinese Medicine),to publish significant case report, and to provide both perspectives on personal experiences in medicine and reviews of the current state of biology and medicine.