O型口蹄疫病毒结构蛋白基因VP1的克隆与原核表达

Cloning and Prokaryotic Expression of VP1 Gene of Foot-and-Mouth Disease Virus(FMDV) Type O

[目的]为进一步研究口蹄疫病毒的诊断方法提供理论依据。[方法]根据GenBank上已公布的O型口蹄疫病毒核苷酸序列,设计合成1对特异性引物,通过RT-PCR扩增VP1基因,将其克隆至表达载体pGEX-6p-1中,经测序鉴定目的基因正确地整合至表达质粒中,用IPTG诱导重组基因表达。[结果]通过PCR扩增获得682 bp的目的片段,所克隆的VP1基因在原核细胞中成功表达,表达产物为融合蛋白,经SDS-PAGE鉴定分子量为51 kD,Western Blot结果表明融合蛋白具有免疫原性。[结论]获得了猪O型口蹄疫VP1融合蛋白,可作为包被抗原用于口蹄疫诊断试剂盒的研制。

[ Objective] To provide theoretical basis for further investigation on diagnosis method of FMDV. [ Method ] According to the nucleotide sequence of foot-and-mouth disease virus （FMDV） type O pubhshed in GenBank, a pair of special primers were designed to amplify VP1 gene by RT-PGR and the products were subsequently inserted into the expression vector pGEX-6p-1. With sequencing, the target gene was correctly cloned into the expression vector, which was induced by IPTG to express the recombinant gene. [ Result] The target gene, about 682 bp, was obtained by PCR and the cloned VP1 gene was successfully expressed in prokaryotic cells. The expressed products were fusion proteins with molecular weight 51 kD by SDS-PAGE identification. The resuh of Western Blot indicated the immunogenicity of the fusion proteins. [ Conclusion ] The fusion proteins of VP1 gene of FMDV type O were obtained, which could be used as coating antigen for preparation of foot- and-mouth disease diagnostic kit.