狂犬病病毒ERA株P蛋白的可溶性表达、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】 通过扩增狂犬病病毒ERA株的P基因并定向克隆到原核表达载体pET-32a(+)中,获得阳性P基因重组质粒pET-P,转化原核表达宿主菌BL21(DE3),用IPTG诱导并优化诱导条件,使其在大肠杆菌中以可溶性表达为主。SDS-PAGE电泳分析发现,重组质粒pET-P成功的表达出大小约50 KD的融合蛋白,用Ni-NTA亲和层析在非变性条件下纯化P蛋白,获得纯度较高的蛋白,Western blot鉴定纯化后的P蛋白,结果表明纯化后的P蛋白均可被RV阳性血清和抗His的单克隆抗体识别,证实其免疫原性良好且成功融合表达了His标签蛋白,高纯度P蛋白免疫小鼠制备多克隆抗体,本研究制备的抗体经Westernblot和间接免疫荧光鉴定,都能特异性的识别狂犬病病毒感染细胞中表达的P蛋白,具有较好的特异性,制备的抗P蛋白的抗血清将为进一步研究P蛋白的潜在功能打下良好的基础。更多还原

【Abstract】 P gene of rabies virus ERA strain was amplified and cloned into prokaryotic expression vector pET-32a(+),the recombinant plasmid pET-P was transformed into the E.coli BL21(DE3) cells,and induced with IPTG,and optimized expression condition.SDS-PAGE analysis showed that the recombinant pET-P plasmid successfully expressed P protein of 50 KD.The P protein was mainly expressed in the soluble form,and it was purified by Ni-NTA under native condition,the purified P protein was used to immunize mice and product a polyclonal antibody.Using western blot and IFA assay,the anti-P protein antibody could specific recognize the P protein in rabies virus infected cells.The anti-P protein is a useful for further study on the role of P protein.更多还原