猪细小病毒1型VP2基因的原核表达及反应原性分析

Prokaryotic Expression of Gene VP2 of Porcine Parvovirus Type 1 and the Reactinogenicity Analysis of the Expressed Protein

旨在研究猪细小病毒1型(PPV1)VP2蛋白(第155-439位氨基酸)的抗原性,为开发PPV1的检测方法奠定基础。以PPV1型AV31株的DNA为模板,扩增获得849 bp的目的片段,扩增产物克隆入p ET30a(+)原核表达载体,构建p ET30aPPV1-VP2(155-439 aa)重组质粒,转入大肠杆菌BL21(DE3);在37℃,以1 mmol/L IPTG诱导表达6 h;采用Ni-NTA树脂亲和层析纯化重组蛋白,并用不同浓度的尿素对纯化蛋白进行复性。SDS-PAGE分析表明,该VP2编码基因在大肠杆菌中得到表达,蛋白大小约为39 k D;Western blot检测结果表明,该重组蛋白与PPV1阳性血清发生特异性反应,与NA-PRRSV和PCV2阳性血清不发生交叉反应。该实验成功构建了PPV1-VP2(155-439 aa)原核表达载体,实现了在大肠杆菌中的表达,纯化后的复性蛋白具有较好的反应原性。

This experiment is aimed to study the antigenicity of VP2 protein （ amino acid 155-439 ） of porcine parvovirus type 1 （ PPV1 ） for laying a base for the development of detecting PPV1. The 849 bp target fragment was amplified using the DNA of strain AV31of PPV1 as the template. The product was cloned into pET30a （ ＋ ） vector, and recombinant plasmid pET3Oa-PPV1-VP2 （ amino acid 155-439 ） was constructed, then transferred into Escherichia coli BL21 （ DE3 ） for the 6 h induced expression by IPTG. The recombinant protein was purified by Ni-NTA, and refolded by different concentration of urea. The results of SDS-PAGE showed that the gene VP2 was successfully expressed in E. coli BL21 （ DE3 ） with a relative molecular weight of 39 kD. The results of Western blot showed that this recombined protein specifically reacted with PPV1 positive serum, while no cross reaction with NA-PRRSV and PCV2 positive serum. This study achieved the aims ： the recombinant vector pET30a-PPV1-VP2 was successfully constructed, and the gene was successfully expressed in E. coli, and the purified and re-folded protein demonstrated promising reactinogenicity.