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FMDVRNA,且主要以引物依赖的方式合成病毒基因组。
The 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.
出处
《中国病毒学》
CSCD
2006年第4期375-379,共5页
Virologica Sinica
基金
国家重点基础研究发展规划项目"973"(1999011904)
关键词
口蹄疫病毒
RNA依赖的RNA聚合酶
体外复制实验
荧光定量PCR
Foot-and-mouth disease virus (FMDV)
RNA-dependent RNA polymerase
Replicationsystem in vitro
Strand-specific real-time RT-PCR