高致病性PRRSV JL-04/12株核衣壳蛋白的表达与抗原性分析

Expression and Antigenicity Analysis of Nucleocapsid Protein from Highly Pathogenic PRRSV JL-04/12 Strain

为获得高浓度、高质量的有效原核表达蛋白作为ELISA诊断抗原,通过RT-PCR法扩增猪繁殖与呼吸综合征病毒(PRRSV)JL-04/12株的核衣壳蛋白(N蛋白)基因并克隆到原核表达载体pGEX-6P-1中,转入到大肠埃希菌BL-21感受态细胞,经IPTG诱导表达,通过温度和IPTG浓度的优化,进行重组蛋白的高效表达,经SDS-PAGE和Western blot对表达产物的特性进行分析,利用GST柱纯化表达蛋白,并免疫小鼠,ELISA检测其刺激产生抗体的能力-抗原的免疫原性。结果表明,0.1mmol/L的IPTG、28℃诱导4h可高效诱导重组蛋白的表达,在表达上清中目的蛋白达40%,原核表达的重组N蛋白可诱导小鼠产生抗体,抗体效价为1∶256。原核表达的N蛋白与PRRSV具有相同的抗原性。

In order to obtain high concentration and efficient prokaryotic expression protein as antigen for ELISA diagnosis,the nucleocapsid protein （N protein） gene of porcine reproductive and respiratory syndrome virus （PRRSV） was amplificated by RT-PCR and cloned into prokaryotic expression vector pGEX-6P-1. The cloned products were transformed into Escherichia coli BL-21 competent cells. The expression of recombinant fusion protein was induced by IPTG in E. coli BL21（DE3） system. The efficient expression of recombinant proteins was obtained by the temperature and IPTG concentration optimization. The expressed protein was detected by SDS-PAGE and Western blot, and purified with GST column. The immunogenicity of N protein was examined in mice. The results showed that the expression of recombinant protein was efficiently induced under the condition：0.1 mmol/L IPTG,28℃ and 4 h. The target protein was in the supernatant and accounted for 40% of the total protein. The prokaryotic expressed recombinant N protein can induce the production of antibody in mice,and the antibody titer was 1 ： 256. Prokaryotic expression N protein had the same antigenicity with PRRSV.