非洲猪瘟病毒H240R蛋白的原核表达及多克隆抗体的制备

Prokaryotic Expression of African Swine Fever Virus H240R Protein and Preparation of Polyclonal Antibodies

非洲猪瘟(ASF)是由非洲猪瘟病毒(ASFV)引起的急性、高热、严重出血、致死率极高的动物传染病,给我国养殖业造成严重的经济损失。本研究将非洲猪瘟病毒H240R基因克隆至pET-28a(+)载体中,成功构建了pET-28a(+)-H240R原核表达载体,将重组载体转化入BL21感受态细胞,用IPTG诱导表达。SDS-PAGE和Western blot鉴定结果表明,成功获得了H240R重组蛋白,将其纯化后作为抗原免疫新西兰大白兔,分离血清并分析其反应特异性。ELISA检测结果显示,血清抗体效价达到1∶256000。Western blot和间接免疫荧光(IFA)鉴定结果表明,制备的兔源多克隆抗体能够同时与原核和真核表达的H240R蛋白反应。本研究成功制备针对ASFV H240R蛋白的多克隆抗体,该多克隆抗体具有良好的反应活性及特异性,为建立针对ASFV抗原、抗体的免疫学检测方法提供了新的思路。

African swine fever(ASF)is a highly contagious disease caused by African swine fever virus(ASFV),characterized by high fever,severe bleeding and extremely high fatality rate.The disease causes serious economic losses to the worldwide swine industry.In this study,the ASFV H240R gene was cloned into pET-28a(+)vector to construct the prokaryotic expression vector of pET-28a(+)-H240R.The recombinant vector was then transformed into BL21 competent cells and the expression was induced with IPTG.The recombinant H240R protein was successfully expressed as determined in SDS-PAGE and Western blot and purified protein was used to immunize New Zealand white rabbits.The rabbit serum samples had ELISA titer at 1:256000 and good reactivity with the H240R protein in Western blot and indirect immunofluorescence assay.The availability of rabbit antibodies provided a good tool for the development of detection methods for ASFV.