非洲猪瘟病毒EP364R蛋白在杆状病毒表达系统中的表达及鉴定

Expression and Identifi cation of African Swine Fever Virus EP364R Protein in Baculovirus Expession System

非洲猪瘟病毒(ASFV)EP364R蛋白是ASFV的ERCC4核酸酶结构域。为了获得具有免疫原性的EP364R蛋白,本研究将ASFV EP364R基因构建到杆状病毒表达载体上,将重组质粒转座DH10Bac感受态中,经过3次蓝白斑筛选,获得重组BacmidEP364R质粒,将Bacmid-EP364R质粒转染至细胞中,收获上清,在正常细胞中传三代,以获得重组杆状病毒rBac-EP364R。使用Western blot和间接免疫荧光(IFA)实验鉴定重组蛋白得到表达及其免疫原性。本研究结果显示,在感染的细胞可检测到EP364R蛋白的表达,并可被His单克隆抗体特异性识别。结果表明杆状病毒系统成功表达EP364R蛋白并具有良好的反应活性,为后续开展ASFV血清学诊断和亚单位疫苗的研究奠定基础。

African swine fever virus(ASFV)EP364R protein is the ERCC4 nuclease domain of ASFV.In order to obtain immunogenic EP364R protein.In this study,the ASFV EP364R gene was cloned into baculovirus expression vector,the recombinant plasmid was transposed into DH10Bac competent,after three blue-white screening,the recombinant Bacmid-EP364R plasmid was obtained,and the Bacmid-EP364R plasmid was transfected into cells.In the supernatant was harvested and passed in normal cells for three generations to obtain the recombinant baculovirus rBac-EP364R.Western blotting and indirect immunofluorescence(IFA)experiments were used to identify the expression and immunogenicity of the recombinant protein.The results showed that the expression of EP364R protein could be detected in the infected cells and can be specifically recognized by anti-His monoclonal antibody.The results show that the baculovirus system successfully expresses EP364R protein and has good reactivity,which lays the foundation for the subsequent development of ASFV serological diagnosis and subunit vaccine research.