狂犬病毒ERA株基质蛋白的原核表达、纯化及多克隆抗体的制备

【作者】 孟先明； 潘艳； 蔡新斌； 陆专灵； 梁湘； 唐海波； 杨剑； 张红普； 罗廷荣；

【机构】 广西大学动物科学技术学院； 广西亚热带生物资源保护利用重点实验室；

【摘要】 应用PCR扩增狂犬病病毒ERA株的M基因,定向插入到原核表达载体pET-32a(+)中,把构建的重组质粒pET-M转化原核表达宿主菌株BL21(DE3),通过IPTG诱导和优化表达条件,在大肠杆菌获得高效表达。重组M蛋白主要以包涵体的形式表达,用含不同浓度尿素的包涵体洗涤液处理包涵体,纯化M蛋白,用纯化的M蛋白免疫小鼠制备多克隆抗体,经过Western blot和间接免疫荧光试验证实抗M蛋白的血清能特异地识别狂犬病病毒感染细胞中表达的M蛋白,为进一步研究狂犬病病毒M蛋白的功能奠定基础。更多还原

【Abstract】 M gene of rabies virus ERA strain was amplified and inserted into prokaryotic expression vector pET-32a(+),the recombinant plasmid pET-M was transformed into the E.coli BL21(DE3) cells,and induced by IPTG,the expression condition was optimized and M protein was expressed in a high level.The M protein was mainly expressed in a form of inclusion body,was well purified with different concentration of urea buffer.The purified M protein was used to immunize mice and product polyclonal antibody,it may provide a useful tools for the further study of the role of M protein.Both Western blot and indirect immunofluorescence assay confirmed the polyclonal antibody could recognize the native M protein in rabies virus infected cells.更多还原