Role of ORF7 in the Genome of SARS-CoV

ZHAO Lin; TU Yi-xian; YU Yang; JIN Hua-li; CHEN Ze; WANG Bin

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August 2006

ZHAO Lin, TU Yi-xian, YU Yang, JIN Hua-li, CHEN Ze and WANG Bin. Role of ORF7 in the Genome of SARS-CoV[J]. Virologica Sinica, 2006, 21(4): 343-347.

Citation: ZHAO Lin, TU Yi-xian, YU Yang, JIN Hua-li, CHEN Ze, WANG Bin. Role of ORF7 in the Genome of SARS-CoV .VIROLOGICA SINICA, 2006, 21(4) : 343-347.

SARS冠状病毒基因结构中未知蛋白Orf7的研究

赵琳 ¹ ,
涂亦娴 ¹ ,
于杨 ¹ ,
金华利 ¹ ,
陈则 ² ,
王宾 ^1*

1.
1.中国农业大学农业生物技术国家重点实验室和生物学院微生物与免疫学系，北京100094
2.
中国科学院武汉病毒研究所，湖北武汉，430071

摘要

为探讨SARS-CoV未知蛋白在基因表达调控中的作用，本研究以鉴定的SARS-CoV BJ01毒株cDNA为材料，采用PCR方法扩增SARS-CoV未知蛋白基因Orf7，Orf8，Orf9，引入GFP和pDsRed作为报告基因，构建出5种表达质粒：pEGFP-Orf7，pEGFP-Orf8，pEGFP-Orf79，pcDNA3-Orf7，pcDNA3-Orf8。进行体外转染实验后，采用荧光酶标仪测量其细胞中荧光蛋白的表达量，再采用流式细胞仪进一步进行瞬时表达的分析比较。结果发现，pcDNA3-Orf7与pEGFP共转染时，pcDNA3-Orf7能够增强绿色荧光蛋白(GFP)的表达，pEGFP-Orf7与pDsRed共转染时pEGFP-Orf7可以增强红色荧光蛋白(RFP)的表达, 且同时表达红色和绿色荧光的细胞数增加。本文首次证明了ORF7编码的63个氨基酸的蛋白具有反式激活作用，即为SARS-CoV自身的反式激活因子。
- SARS冠状病毒（SARS-CoV）
- , 反式激活因子
- , ORF7
- , ORF8
- , ORF9

Role of ORF7 in the Genome of SARS-CoV

ZHAO Lin ¹ ,
TU Yi-xian ¹ ,
YU Yang ¹ ,
JIN Hua-li ¹ ,
CHEN Ze ² ,
WANG Bin ^1*

1.
1.State Key Laboratory for Agro-Biotechnology and Department of Microbiology and Immunology, College of Biological Science, China Agricultural University, Beijing 100094, China
2.
Wuhan Institute of Virology, Chinese Academy of Sciences, Wuhan 430071, China

Abstract

The aim of this study was to characterize functions of the unknown open-reading frames, Orf7, Orf8 and Orf9 within the Severe acute respiratory syndrome coronavirus (SARS-CoV) genome. We cloned the cDNAs of Orf7, Orf8, Orf9 from the SARS-CoV BJ01 strain were cloned by RT-PCR and constructed into five expression vectors designated as pEGFP-Orf7, pEGFP-Orf8, pEGFP-Orf79, pcDNA3-Orf7 and pcDNA3-Orf8. Hela cells were transfected with the constructs and analyzed for transient expressions by fluorescent analysis. We observed that the expression of pEGFP was enhanced when pcDNA3-Orf7 and pEGFP were co-transfected into Hela cells. Also the expression of pDsRed was increased with pEGFP-Orf7 and pDsRed. More cells which expressed green and red fluorescence at the same time were detected. Based on these results, we demonstrated for the first time that the 63 amino acid protein encoded by Orf7 served as a transcriptional transactivator in SARS-CoV.
- SARS-CoV
- , Transcriptional transactivator
- , ORF7
- , ORF8
- , ORF9

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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

Role of ORF7 in the Genome of SARS-CoV

1. 1.State Key Laboratory for Agro-Biotechnology and Department of Microbiology and Immunology, College of Biological Science, China Agricultural University, Beijing 100094, China
2. Wuhan Institute of Virology, Chinese Academy of Sciences, Wuhan 430071, China

Abstract: The aim of this study was to characterize functions of the unknown open-reading frames, Orf7, Orf8 and Orf9 within the Severe acute respiratory syndrome coronavirus (SARS-CoV) genome. We cloned the cDNAs of Orf7, Orf8, Orf9 from the SARS-CoV BJ01 strain were cloned by RT-PCR and constructed into five expression vectors designated as pEGFP-Orf7, pEGFP-Orf8, pEGFP-Orf79, pcDNA3-Orf7 and pcDNA3-Orf8. Hela cells were transfected with the constructs and analyzed for transient expressions by fluorescent analysis. We observed that the expression of pEGFP was enhanced when pcDNA3-Orf7 and pEGFP were co-transfected into Hela cells. Also the expression of pDsRed was increased with pEGFP-Orf7 and pDsRed. More cells which expressed green and red fluorescence at the same time were detected. Based on these results, we demonstrated for the first time that the 63 amino acid protein encoded by Orf7 served as a transcriptional transactivator in SARS-CoV.