Laboratory for Molecular Virology, Great Lakes Forestry Centre, 1219 Queen St E, Sault Ste Marie, ON P6A 2E5, Canada.
J Virol Methods. 2010 Apr;165(1):1-8. doi: 10.1016/j.jviromet.2009.05.015. Epub 2009 May 27.
Spheroidin (SPH) is the most abundant late protein in cells infected with the Amsacta moorei entomopoxvirus (AMEV). This locus can be used for expression of exogenous genes because it is not essential for virus replication. The sph promoter contains a conserved TAAATG motif, which serves as the site of initiation for both transcription and translation. Additional sequences downstream of the conserved motif have been shown to be involved in high-level expression of the sph gene. As a first step towards developing a protein expression vector based on the sph locus, four recombinant AMEV viruses expressing either gfp or lacZ were constructed. Both reporter genes were expressed under the control of the sph promoter containing the TAAATG motif. An additional 6 bp or 21 bp of sph coding region was included in three of the recombinants, to be expressed as an N-terminal fusion protein of GFP or LacZ. GFP and beta-galactosidase expression was observed at 2 days post-infection and continued throughout the observation period. The highest level of reporter gene expression was observed in the recombinant containing 21 bp from the sph coding region. These results indicate that sph locus of AMEV can be used successfully to express exogenous genes.
球形体蛋白（SPH）是感染 Amsacta moorei 昆虫痘病毒（AMEV）的细胞中最丰富的晚期蛋白。该基因座可用于外源基因的表达，因为它对病毒复制不是必需的。sph 启动子含有一个保守的 TAAATG 基序，该基序既是转录和翻译的起始位点。已证明保守基序下游的额外序列参与 sph 基因的高水平表达。为了基于 sph 基因座开发蛋白质表达载体，首先构建了四种表达 GFP 或 lacZ 的重组 AMEV 病毒。这两个报告基因都受包含 TAAATG 基序的 sph 启动子的控制。在三种重组体中，包含 6 个碱基或 21 个碱基的 sph 编码区被包含，以 GFP 或 LacZ 的 N 端融合蛋白的形式表达。在感染后 2 天观察到 GFP 和β-半乳糖苷酶的表达，并在整个观察期内持续表达。在包含来自 sph 编码区的 21 个碱基的重组体中观察到最高水平的报告基因表达。这些结果表明 AMEV 的 sph 基因座可成功用于表达外源基因。
