FMDV 3D Gene Cloning Expression and Functional Analysis

ZHANG Qian; GU Chao-jiang; SHI Li-li; LI Yong; QU San-fu; ZHENG Cong-yi*

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August 2006

ZHANG Qian, GU Chao-jiang, SHI Li-li, LI Yong, QU San-fu and ZHENG Cong-yi*. FMDV 3D Gene Cloning Expression and Functional Analysis[J]. Virologica Sinica, 2006, 21(4): 375-379.

Citation: ZHANG Qian, GU Chao-jiang, SHI Li-li, LI Yong, QU San-fu, ZHENG Cong-yi*. FMDV 3D Gene Cloning Expression and Functional Analysis .VIROLOGICA SINICA, 2006, 21(4) : 375-379.

口蹄疫病毒3D基因的克隆表达及功能分析

1.
武汉大学生命科学学院病毒学国家重点实验室，湖北武汉 430072

摘要

利用RT-PCR技术扩增了口蹄疫病毒(FMDV)编码RNA依赖的RNA聚合酶的3D基因，并将其克隆到原核表达质粒载体pET-28a(+)中。3D基因经测序确认后在大肠杆菌BL-21中表达，表达产物纯化的目的蛋白进行Western-blotting检测，获得分子量约55KDa的单一3D基因表达产物。利用RNA体外复制体系和荧光定量PCR技术，证明纯化的3D基因表达产物RNA依赖的RNA聚合酶具有较高的酶活性，可以在体外从头合成FMDV RNA，且主要以引物依赖的方式合成病毒基因组。
- 口蹄疫病毒
- , RNA依赖的RNA聚合酶
- , 体外复制实验
- , 荧光定量PCR

FMDV 3D Gene Cloning Expression and Functional Analysis

1.
State Key Laboratory of Virology, College of Life Sciences, Wuhan University, Wuhan 430072, China

Abstract

he RdRp gene of Foot-and-mouth disease virus was amplified by reverse transcription polymerase chain reaction (RT-PCR) using pfuultraTM High-Fidelity DNA polymerase, and was then cloned into the prokaryotic expression vector pET-28a (+). E.coli BL-21 cells were transformed with the recombinant plasmid and the target protein was expressed in soluble form. The purified recombinant protein was confirmed to be expressed correctly by Western-blotting. The RNA in vitro replication assay was used to determine the high activity of purified RdRp. The product of replication system was quantified by strand-specific real-time RT-PCR. These results suggested that the purified RdRp could initiate the de-novo RNA synthesis but was mainly in a primer-dependent manner, which shows high activity of RNA-dependent RNA polymerase. The Vpg protein primer can dramatically improve the capacity of RNA synthesis.
- Foot-and-mouth disease virus (FMDV)
- , RNA-dependent RNA polymerase
- , Replication system in vitro
- , Strand-specific real-time RT-PCR

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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

FMDV 3D Gene Cloning Expression and Functional Analysis

1. State Key Laboratory of Virology, College of Life Sciences, Wuhan University, Wuhan 430072, China

Abstract: he RdRp gene of Foot-and-mouth disease virus was amplified by reverse transcription polymerase chain reaction (RT-PCR) using pfuultraTM High-Fidelity DNA polymerase, and was then cloned into the prokaryotic expression vector pET-28a (+). E.coli BL-21 cells were transformed with the recombinant plasmid and the target protein was expressed in soluble form. The purified recombinant protein was confirmed to be expressed correctly by Western-blotting. The RNA in vitro replication assay was used to determine the high activity of purified RdRp. The product of replication system was quantified by strand-specific real-time RT-PCR. These results suggested that the purified RdRp could initiate the de-novo RNA synthesis but was mainly in a primer-dependent manner, which shows high activity of RNA-dependent RNA polymerase. The Vpg protein primer can dramatically improve the capacity of RNA synthesis.