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.

FMDV 3D Gene Cloning Expression and Functional Analysis

  • Available online: 20 July 2006
  • 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.

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    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.

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