猪繁殖与呼吸综合征病毒Hn-1/06株GP3蛋白的原核表达与纯化

Prokaryotic Expression of GP3 Protein of Porcine Reproductive and Respiratory Syndrome Virus Hn-1/06 Strain

根据GenBank中PRRSV美洲株ATCC VR2332的ORF3基因序列,利用Primer 5.0软件设计合成了1对特异性引物,经RT-PCR从河南分离株Hn-1/06株扩增得到了大小为765 bp的片段,并将扩增的片段插入pTG19-T载体进行测序而获得含有ORF3的阳性克隆pTG19-T-GP3。将此基因亚克隆到原核表达载体pET-32a,经筛选获得了阳性重组质粒pET-GP3,进而在IPTG的诱导下成功表达,获得了大小约为45 kDa的融合蛋白GP3-His,纯化后经Western blot检测证实表达的融合蛋白具有良好的生物学活性。从而为进一步研究PRRSV次要结构蛋白GP3的免疫特性和功能奠定了基础。

One pair of specific primers was designed according to the sequence of PRRSV ORF3 gene published in GenBank （Accession number PRU87392）, and was used to amplify the ORF3 gene from a PRRSV Hn-1/06 strain by RT-PCR method, resulting in a 765 bp gene fragment. The RT-PCR product was directly cloned into the pTG19-T vector and the recombinant plasmid was identified by enzyme digesting and DNA sequencing. The positive clone was designated as pTG19-T-GP3 .After being cloned into the prokaryotic expression vector pET-32a, the ORF3 gene was successfully obtained by the induction of IPTG. Western-blot test confirmed that the expressed fusion protein could specifically react with the antiserum against PRRSV. It could be used for the further investigation on immunogenicity and function of GP3 of PRRSV.