【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。更多还原

【Abstract】 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.更多还原