人冠状病毒 OC43 核衣壳蛋白的杆状病毒表达及用于检测人抗人冠状病毒 OC43 抗体的蛋白质印迹检测方法的建立
Baculovirus expression of HCoV-OC43 nucleocapsid protein and development of a Western blot assay for detection of human antibodies against HCoV-OC43.
作者信息
Mourez Thomas, Vabret Astrid, Han Yang, Dina Julia, Legrand Loïc, Corbet Sandrine, Freymuth François
机构信息
Laboratory of Human and Molecular Virology, University Hospital of Caen, Avenue Georges Clemenceau, 14 033 Caen Cedex, France.
出版信息
J Virol Methods. 2007 Feb;139(2):175-80. doi: 10.1016/j.jviromet.2006.09.024. Epub 2006 Oct 31.
Abstract
The nucleocapsid (N) gene of human coronavirus strain OC43 (HCoV-OC43) was amplified by reverse transcriptase-polymerase chain reaction, and cloned in pENTR/D-TOPO plasmid. This plasmid containing the N gene was recombined with in a BaculoDirect 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.
摘要
通过逆转录聚合酶链反应扩增人冠状病毒OC43株（HCoV-OC43）的核衣壳（N）基因，并克隆到pENTR/D-TOPO质粒中。将含有N基因的该质粒与为表达与含有V5表位的C末端多组氨酸标签融合的N蛋白而设计的BaculoDirect杆状病毒DNA进行重组。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和蛋白质免疫印迹法进行分析，并通过Ni2+亲和程序进行纯化。使用纯化的重组N蛋白，通过蛋白质免疫印迹试验检测了100名医护人员和5名2至3岁儿童的血清。所有成人血清和两份儿童血清检测结果呈阳性。
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