【摘要】 将猪源O型口蹄疫病毒(Foot-and-mouthdiseasevirus,FMDV)VP1基因克隆到原核表达载体pMBX上,成功地构建重组表达质粒pMBX-VP1。将其转化大肠杆菌(Escherichiacoli)BL21(DE3)感受态细胞中,经SDS-PAGE分析,融合蛋白分子量约为58kD,表达产物占菌体总蛋白的35.5%。经蛋白可溶性分析,目的蛋白在裂解沉淀中占6.8%,在裂解上清中占31.2%,融合蛋白绝大部分是以可溶形式表达的。经Westernblot证实,可溶表达的融合蛋白与FMDV阳性血清具有良好的免疫反应性。将可溶表达的融合蛋白用50%Ni-NTA树脂纯化,用融合蛋白作包被抗原,通过ELISA方法,能特异性地检测出口蹄疫阳性血清。更多还原

【Abstract】 Foot-and-mouth disease virus (FMDV) VP1 gene was amplified by RT-PCR. The VP1 fragment was inserted into plasmid pMBX to obtain recombinant plasmid pMBX-VP1. The pMBX-VP1 was transformed into Escherichia coli BL-21(DE3) to induce VP1 gene fusion expression with 1 mmol/L Isopropylthio-β-D-galactoside (IPTG). The fusion protein band with molecular weight of 58 kD was visible on the SDS-PAGE gel. The density scanning showed that the largest amount of the fusion protein was 35.5% of total bacterial protein. The amounts of the soluble form of the fusion in supernatant of lysate was up to 31.2%, and 6.8% in sediment. Western blot result shawed that the expression products could specifically reacted with the antisera against FMDV. The soluble fusion protein was purified by 50%Ni-NTA affinity chromatography and used as an antigen, and FMDV antibody could be detected by ELISA method.更多还原