猪繁殖与呼吸综合征病毒GP5蛋白的表达及间接ELISA抗体检测方法的建立

Expression of GP5 protein of porcine reproductive and respiratory syndrome virus and establishment of the indirect ELISA antibody detection assay

为建立检测感染猪繁殖与呼吸障碍综合症病毒(PRRSV)或PRRSV弱毒疫苗免疫后猪群体内抗体水平的方法,本研究以PRRSV GP5基因的原核表达蛋白为包被抗原,建立了间接ELISA抗体检测方法。利用RT-PCR扩增PRRSV GP5基因,将其克隆于p GEX-6p-1中构建原核重组表达载体p GEX-6P-GP5,转入大肠杆菌BL21(DE3)中进行诱导表达,以梯度尿素法对表达蛋白纯化后,以其为包被抗原,利用方阵试验对间接ELISA方法进行优化,最终确定了GP5蛋白的最佳包被浓度是150 ng/孔,被检血清的最佳稀释倍数为80倍。试验结果显示,该检测方法的特异性、敏感性、重复性很强。从山东各地采集203份猪血清样品进行检测,结果显示该检测方法与商品化PRRSV抗体检测试剂盒(法国LSI)检测结果的符合率为96.5%。本研究建立的间接ELISA方法,为猪群体内PRRSV抗体水平的检测提供了重要的检测手段,在疫苗免疫效果的评价方面有着广泛的应用。

To detect the antibody in pigs after PRRSV infection and PRRS vaccine immunization, the indirect ELISA antibody detection assay was established based on GP5 prokaryotic expression protein of PRRSV as coating antigen in this study. PRRSV GP5 gene was amplified by RT-PCR and cloned into pGEX-6p-1 to construct pEGX-6P-GP5. pEGX-6P-GP5 was transformed into BL21 （DE3） and induced, then the recombinant GP5 protein was expressed. The indirect ELISA was established with the punfied GP5 protein as the coating antigen, and was optimized by square test. The optimization results showed the coating concentration of GP5 protein was 150ng/cell and the detected serum was diluted by 80 times. The specificity, sensitivity and reproducibility of the assay were very high. 203 serum samples collected from Shandong province were detected by the indirect ELISA assay, the coincidence was 96.5% between the indirect ELISA this study and the ELISA detection Kit （France LSI）. The indirect ELISA method established in this study was an important detection assay for PRRSV antibody levels in pig groups and would have been widely used in detection of vaccine immune effect.