Expression of TT Virus ORF2 and the Protein Localization of VP2 in Cos7 Cells

HE Yan; YE Lin-bai*; LI Li; LIAO Qing-jiao; Khalid A Timani; SHE Ying-long; YE Li; WU Zheng-hui

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October 2004

HE Yan, YE Lin-bai*, LI Li, LIAO Qing-jiao, Khalid A Timani, SHE Ying-long, YE Li and WU Zheng-hui. Expression of TT Virus ORF2 and the Protein Localization of VP2 in Cos7 Cells[J]. Virologica Sinica, 2004, 19(5): 431-434.

Citation: HE Yan, YE Lin-bai*, LI Li, LIAO Qing-jiao, Khalid A Timani, SHE Ying-long, YE Li, WU Zheng-hui. Expression of TT Virus ORF2 and the Protein Localization of VP2 in Cos7 Cells .VIROLOGICA SINICA, 2004, 19(5) : 431-434.

TT病毒ORF2在Cos7细胞中的表达及蛋白质定位

1.
武汉大学生命科学院病毒研究所，教育部病毒学重点实验室，湖北武汉430072

通讯作者： 叶力, ;

摘要

应用PCR方法从含有TT Virus ORF2的质粒pET-His- TTV2中扩增出606bp的蛋白质编码区，并将其克隆到真核表达载体pEGFP-N1中以表达成GFP-VP2融合蛋白。构建出的重组质粒pEGFPTTV2经过酶切分析和PCR鉴定。用脂质体介导法将pEGFPTTV2质粒DNA转染Cos7细胞，通过RT-PCR分析，证实细胞中存在ORF2基因的转录产物。用共聚焦显微镜结合PI染色技术研究TTV VP2蛋白在细胞中的分布情况。结果表明，TTV VP2分布在细胞质中和细胞核膜内侧。因此推测VP2作为一种非结构蛋白，功能可能是参与病毒DNA的复制或转录。
- 输血传播病毒（TTV）ORF2
- , GFP
- , VP2
- , PI
- , 蛋白质定位

Expression of TT Virus ORF2 and the Protein Localization of VP2 in Cos7 Cells

1.
Key lab of Virology, Institute of Virology, College of life Sciences, Wuhan University, Wuhan 430072, China

Corresponding author: YE Li,

Abstract

Coding sequences of Transfusion transmiffed virus(TT virus or TTV) ORF2 was amplified by PCR, using plasmid pET-His-TTV2 containing the full-length ORF2 of TTV as template. The amplified fragment of 606bp was cloned into plasmid pEGFP-N1 and GFP-VP2 fusion protein was expressed. The recombinant plasmid pEGFPTTV2 was identified by restriction enzyme analysis and PCR. The pEGFPTTV2 was transfected into Cos7 cell line using LipoFectamine 2000, and the transcription products of TTV ORF2 gene in the transfected cells were confirmed by RT-PCR. Localization of the fusion protein was detected by confocal microscope, using PI to visualize the nuclear DNA. The result showed that VP2 was localized in cytoplasm and the inner nuclear membrane. Thus, VP2, as a non- structure protein, may play a role during DNA replication or transcription of TT virus.
- TTV ORF2
- , GFP
- , VP2
- , PI
- , Protein Localization

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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

Expression of TT Virus ORF2 and the Protein Localization of VP2 in Cos7 Cells

Corresponding author: YE Li,

1. Key lab of Virology, Institute of Virology, College of life Sciences, Wuhan University, Wuhan 430072, China

Abstract: Coding sequences of Transfusion transmiffed virus(TT virus or TTV) ORF2 was amplified by PCR, using plasmid pET-His-TTV2 containing the full-length ORF2 of TTV as template. The amplified fragment of 606bp was cloned into plasmid pEGFP-N1 and GFP-VP2 fusion protein was expressed. The recombinant plasmid pEGFPTTV2 was identified by restriction enzyme analysis and PCR. The pEGFPTTV2 was transfected into Cos7 cell line using LipoFectamine 2000, and the transcription products of TTV ORF2 gene in the transfected cells were confirmed by RT-PCR. Localization of the fusion protein was detected by confocal microscope, using PI to visualize the nuclear DNA. The result showed that VP2 was localized in cytoplasm and the inner nuclear membrane. Thus, VP2, as a non- structure protein, may play a role during DNA replication or transcription of TT virus.