口蹄疫病毒3D基因的真核表达及其表达产物的功能分析

Eukaryotic expression of foot-and-mouth disease virus 3D gene and functional analysis of the expressed product

利用RT-PCR技术扩增口蹄疫病毒(FMDV)编码RNA依赖的RNA聚合酶的3D基因,经测序确认后将其克隆到真核表达质粒载体pcDNA3.1(+)中,重组质粒pcDNA3.1(+)-3D经HindⅢ、XbaⅠ双酶切后转染至BHK-21细胞,用纯化的目的蛋白进行Western-blotting鉴定,并预测该3D基因表达产物的三级结构。结果显示,获得分子质量约55 ku的单一3D基因表达产物,其三级结构较为保守。利用RNA细胞内复制体系和荧光定量RT-PCR技术,证明3D基因表达产物在细胞内可促进口蹄疫病毒基因组的复制。

The 3D gene encoding RNA-dependent RNA polymerase（RdRp） of foot-and-mouth disease virus was amplified by RT-PCR using pfu ultra^TM High Fidelity DNA polymerase, and the amplicon was cloned into the eukaryotic expression vector pcDNA3.1 （＋） to construct recombinant expression plasmid. Then the constructed recombinant plasmid was sequenced and transfected into BHK-21 cells. Western-blotting analysis confirmed that the purified recombinant protein RdRp was expressed correctly in soluble form. Tertiary structure of the expressed RdRp was predicted to be conservative. Activity of the protein was determined by the genomic RNA replication assay in BHK-21 cells. The RNA product of the replication system in cells was quantified by strand-specific real time RT-PCR. The results suggested that the RdRp could dramatically improve the synthesis of RNA.