猪捷申病毒3D蛋白的原核高效表达及其反应活性

根据猪捷申病毒(PTV)Swine/CH/IMH/03株核苷酸序列,设计了1对特异性引物,以全长基因组重组质粒pSK-PTVFL为模板扩增了3D基因,并将扩增产物定向克隆到原核表达载体pET30a(+)中,阳性质粒转化BL21(DE3),阳性菌经0.3 mmol/L IPTG诱导后,进行Western-blotting检测。结果显示,3D蛋白获得了高效表达,表达量占菌体总蛋白的66.1%,且重组蛋白能与PTV Swine/CH/IMH/03株阳性血清发生特异性反应。表明,3D蛋白能在大肠杆菌中高效表达,并具有良好的免疫原性,这为开发相应的鉴别诊断技术奠定了基础。

High-level expression of porcine teschovirus 3D protein gene in Escherichia coli and analysis of its biological activity

According to the nucleotide sequence of porcine teschovirus(PTV) Swine/CH/IMH/03 strain,a pair of specific primers was designed to amplify 3D gene(1 356 bp) from the recombinant plasmid pSK-PTVFL containing the full-length genome of PTV.Then the PCR product was subcloned into pET30a(+) vector.The recombinant plasmid was transformed into Escherichia coli BL21(DE3),and induced with 0.3 mmol/L IPTG.The expression product from the gene was detected by Western-blotting.3D gene was expressed and the expressed protein accounted for 60% of total bacterial proteins,and the expressed protein could be recognized specifically by PTV Swine/CHI/IMH/03 positive serum.The results demonstrated that the 3D protein gene could be expressed efficiently in E.coli and the expressed product showed good immunogenicity.The results provided the basis for the development of diagnostic technique for PTV.