水貂肠炎病毒VP2基因的原核表达及间接ELISA方法的建立

Prokaryotic expression of mink enteritis virus VP2 gene and establishment of indirect ELISA

为了对水貂肠炎病毒(MEV)疫苗免疫后抗体水平的监测和疫苗免疫效力的评价,本研究在分析MEV VP2蛋白抗原性的基础上,设计1对特异性引物克隆VP2蛋白抗原性较好的基因片段,将克隆的基因片段定向插入到pProEXHTb原核表达载体中,构建了VP2基因原核表达重组质粒pProEXHTb-VP2A,并实现在大肠杆菌BL21(DE3)中高效表达,经鉴定表达的重组蛋白以包涵体形式存在,免疫印迹试验表明获得的重组蛋白具有与抗体较好的反应原性。应用His·Bind亲和层析柱纯化重组蛋白VP2A,以纯化后的重组蛋白作为ELISA诊断抗原,并对反应条件进行优化,初步建立了检测MEV抗体的VP2A-ELISA方法。确定了抗原最佳包被浓度为9.65μg/mL,血清最佳稀释倍数为1∶10。判定标准为S/P值≥0.312为阳性,S/P值≤0.243为阴性,介于两者之间为疑似。该抗原不与犬瘟热病毒、阿留申病毒阳性血清反应,具有良好的特异性。采用VP2A-ELISA对180份水貂血清样品进行检测,结果显示VP2A-ELISA与HI试验的符合率达到87.8%,表明建立的间接ELISA方法具有较高的敏感性和特异性,为现地免疫貂群抗体检测和MEV流行病学调查提供了一种简便的血清学诊断方法。

The gene fragment encoding high antigenic domain of mink enteritis virus （MEV） VP2 protein was amplified by PCR from MEV genome and cloned into pProEXHTb vector. The recombinant plasmid was transformed into E. coli BL21 and protein expression analyzed by SDS-PAGE and western blot. The expressed protein was about 24.0 ku and able to specifically react with anti-MEV sera. Indiect ELISA was established to detect antibody against MEV with the purified recombinant VP2A protein as the coating antigen. The assay was specific and showed no cross reaction with antibodies of CDV and ADV. Test on 180 sera by VP2A-ELISA and HI assay showed an agreement ratio of 87.8 % between the two methods. The application of indirect ELISA assay would provide a simple and rapid method of detecting MEV infection or assessing vaccination in the field.