Baculovirus expression of HCoV-OC43 nucleocapsid protein and development of a Western blot assay for detection of human antibodies against HCoV-OC43
Baculovirus expression of HCoV-OC43 nucleocapsid protein and development of a Western blot assay for detection of human antibodies against HCoV-OC43
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DOI：
10.1016/j.jviromet.2006.09.024
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发表时间：
2007-02-01
影响因子：
3.1
通讯作者：
Freymuth, Francois
中科院分区：
文献类型：
--
作者：
Mourez, Thomas;Vabret, Astrid;Freymuth, Francois
通过逆转录酶-聚合酶链式反应扩增人冠状病毒株 OC43 (HCoV-OC43) 的核衣壳 (N) 基因，并克隆到 pENTR (TM)/D-TOPO (R) 质粒中。该含有N基因的质粒与BaculoDirect(TM)杆状病毒DNA重组，该杆状病毒DNA被设计用于表达与含有V5表位的C端多组氨酸标签融合的N蛋白。用重组杆状病毒DNA转染Sf21细胞。从感染细胞中提取重组 N 蛋白，通过 SDS-PAGE 和蛋白质印迹进行分析，并通过 Ni2+ 亲和程序进行纯化。使用纯化的重组 N 蛋白，通过蛋白质印迹法对 100 名医护人员和 5 名 2-3 岁儿童的血清进行了测试。所有成人血清和两份儿童血清均呈阳性结果。 (c) 2006 Elsevier B.V. 保留所有权利。
The nucleocapsid (N) gene of human coronavirus strain OC43 (HCoV-OC43) was amplified by reverse transcriptase-polymerase chain reaction, and cloned in pENTR (TM)/D-TOPO (R) plasmid. This plasmid containing the N gene was recombined with in a BaculoDirect (TM) baculovirus DNA designed in order to express N protein in fusion with a C-terminal polyhistidine tag containing V5 epitope. Sf21 cells were transfected with recombinant baculovirus DNA. Recombinant N protein was extracted from infected cells, analysed by SDS-PAGE and Western blot, and purified by Ni2+ affinity procedure. Sera from 100 healthcare workers and five 2-3-year-old children were tested in a Western blot assay using the purified recombinant N protein. All of the sera from adults and two of the sera from children have a positive result. (c) 2006 Elsevier B.V. All rights reserved.