Cloning and Expression of SARS Coronavirus M gene

YAN Hui-jun; ZHAO Wei; FANG Dan-yun; ZHOU Jing-jiao; LONG Bei-guo; ZHANG Wen-bing; GUO Hui-yu; JIANG Li-fang

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February 2005

YAN Hui-jun, ZHAO Wei, FANG Dan-yun, ZHOU Jing-jiao, LONG Bei-guo, ZHANG Wen-bing, GUO Hui-yu and JIANG Li-fang. Cloning and Expression of SARS Coronavirus M gene[J]. Virologica Sinica, 2005, 20(1): 1-4.

Citation: YAN Hui-jun, ZHAO Wei, FANG Dan-yun, ZHOU Jing-jiao, LONG Bei-guo, ZHANG Wen-bing, GUO Hui-yu, JIANG Li-fang. Cloning and Expression of SARS Coronavirus M gene .VIROLOGICA SINICA, 2005, 20(1) : 1-4.

SARS冠状病毒M基因的克隆及其表达

晏辉钧 ¹ ,
赵卫 ² ,
方丹云 ¹ ,
周经姣 ¹ ,
龙北国 ² ,
张文炳 ² ,
郭辉玉 ¹ ,
江丽芳 ^1*

1.
1.广东广州中山大学基础医学院微生物学教研室 2.南方医科大学热带卫生学系微生物学教研室

摘要

从SARS冠状病毒(GD322株)组织培养上清中提取RNA,进行RT PCR扩增其M基因并克隆到巴氏毕赤酵母表达载体pPICZαB,电穿孔法将重组体整合入酵母菌P. pastoris,经抗生素Zeocin筛选产生的转化子进行表型鉴定,取Mut+菌用甲醇诱导表达,SDS PAGE检测其表达产物。Mut+ 酵母转化菌经甲醇诱导可分泌表达约65kDa和42 kDa的蛋白质,与SARS恢复期病人血清的免疫印迹证实它们为特异的重组M蛋白质,且获得的重组M蛋白质具有免疫反应性。
- SARS冠状病毒
- , M基因
- , 克隆
- , 巴氏毕赤酵母
- , 表达

Cloning and Expression of SARS Coronavirus M gene

Abstract

The full-length gene of membrane protein ( M )of SARS coronavirus (SARS-CoV) was cloned using RT-PCR from the Vero-E6 cells infected with SARS-CoV (GD322 strain) and inserted into the multi-cloning site of the pPICZαB vector. The recombinant plasmid was transtormed into P. pastoris strain X-33 by electroporation and selected by Zeocin. Mut phenotype determination was performed on the yeast transformants and then expression of Mut~+ colonies were induced by methanol. The expressed products were analyzed by SDS-PAGE and Western blotting. Secreted expression was performed by screening Mut~+ colonies in the yeast (transformants). The molecular mass of the recombinant M protein was approximately 65 kDa and 42 kDa and secreted into culture medium when induced with methanol. The expressed protein was able to (react) with SARS convalescence polyclonal antibody.
- SARS coronavirus （SARS-CoV）
- , M gene
- , Clone
- , Pichia pastoris
- , Expression

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通讯作者: 陈斌, bchen63@163.com

1.
沈阳化工大学材料科学与工程学院沈阳 110142

Cloning and Expression of SARS Coronavirus M gene

Abstract: The full-length gene of membrane protein ( M )of SARS coronavirus (SARS-CoV) was cloned using RT-PCR from the Vero-E6 cells infected with SARS-CoV (GD322 strain) and inserted into the multi-cloning site of the pPICZαB vector. The recombinant plasmid was transtormed into P. pastoris strain X-33 by electroporation and selected by Zeocin. Mut phenotype determination was performed on the yeast transformants and then expression of Mut~+ colonies were induced by methanol. The expressed products were analyzed by SDS-PAGE and Western blotting. Secreted expression was performed by screening Mut~+ colonies in the yeast (transformants). The molecular mass of the recombinant M protein was approximately 65 kDa and 42 kDa and secreted into culture medium when induced with methanol. The expressed protein was able to (react) with SARS convalescence polyclonal antibody.