猪繁殖与呼吸综合征病毒JL株Nsp10的表达及鉴定

Expression and Identification for Nsp10 of Porcine Reproductive and Respiratory Syndrome Virus JL Strain

为获得猪繁殖与呼吸综合征病毒JL株非结构蛋白Nsp10,以克隆质粒pMD18-T-Nsp10/JL为模板,将Nsp10基因克隆至原核表达载体pET-32a,转化至大肠杆菌BL21(DE3)中诱导表达,对不同温度和IPTG浓度进行优化,以获得Nsp10蛋白表达的最佳条件,采用SDS-PAGE检测目的蛋白的表达情况,并通过Western blotting检测其免疫活性。结果表明,Nsp10基因成功克隆至pET-32a,有分子量约为43.4 kD的融合蛋白获得表达,重组蛋白于诱导4 h后达到高峰,IPTG浓度为0.2-1.2 mmol/L时,重组蛋白表达量无明显差异,Western blotting证实该表达蛋白具有反应原性。

In order to gain Nspl0 of PRRSV JL strain, the Nsp10 gene was obtained from pMD18-T-Nsp10/JL and inserted into pET- 32a vector, then transformed into Escherichia coli BL21 （ DE3 ） . The expression condition, including temperature and IPTG concentration were optimized. The expressed product was identified by SDS-PAGE and Western blotting. The result showed that Nspl0 gene was cloned into pET-32a successfully. SDS-PAGE confirmed that the molecule weight of induced protein was about 43.4 kD. The expression level of recombinant protein reached a peak after 4 h under IPTG induction and was no significant difference when the concentration of IPTG was 0.2 mmol/L to 1.2 mmol/L. The result displayed a positive reaction by Western blotting analysis.