非洲猪瘟病毒p30蛋白单克隆抗体制备和鉴定

Preparation and Characterization of Monoclonal Antibody Against p30 Protein of African Swine Fever Virus

【目的】制备非洲猪瘟病毒(African swine fever virus,ASFV)p30蛋白的单克隆抗体,为非洲猪瘟快速诊断检测方法的建立和疫苗研究提供材料。【方法】构建p30原核表达质粒pET-30a(+)-p30,转化至大肠杆菌BL21(DE3)感受态细胞中进行诱导表达,通过镍柱亲和层析获得目的蛋白。p30复性后,每2周对BALB/c小鼠免疫3次,将免疫小鼠的脾脏与骨髓瘤细胞融合制备单克隆抗体,通过ELISA方法和亚克隆筛选分泌抗体的杂交瘤细胞。将筛选出的单克隆细胞注射进小鼠腹腔制备腹水,通过Western blotting检测、免疫荧光测定(IFA)和抗体亚型分析来鉴定单克隆抗体。【结果】试验成功构建了p30蛋白的原核表达质粒,并使用IPTG诱导、纯化获得了原核系统表达的重组p30蛋白;成功制备出4株能稳定分泌针对ASFV p30蛋白的单克隆抗体杂交瘤细胞株,分别命名为:1H3B4、3F2F5、6E3A1和9D6B9。经ELISA测定抗体腹水效价在1∶100000~1∶1000000之间。经Western blotting和IFA鉴定筛选得到的单克隆抗体与p30蛋白能发生特异性反应。经亚型鉴定,3F2F5和9D6B92株单克隆抗体的重链均为IgG2b,轻链均为Kappa;6E3A1株单克隆抗体的重链为IgM,轻链为Kappa;1H3B4株单克隆抗体的重链为IgG2b,轻链为Lambda。【结论】试验成功制备了4株针对ASFV p30蛋白的单克隆抗体,并分析了单克隆抗体的免疫学特性,为p30蛋白的生物学功能研究和ASFV血清学检测提供依据。

【Objective】The aim of this experiment was to prepare monoclonal antibody to African swine fever virus(ASFV)p30 protein,and lay the foundation for the establishment of a rapid diagnostic test for ASFV and the research of a vaccine.【Method】The p30 prokaryotic expression plasmid pET-30a(+)-p30 was constructed,transformed into E.coli BL21(DE3)competent cells for induced expression,and then the target protein was obtained by nickel column affinity chromatography.p30 was compounded and immunized to BALB/c mice for three times every two weeks,and the spleen of immunized mice was fused with myeloma cells to prepare monoclonal antibody by ELISA method and subclonal screening of antibody-secreting hybridoma cells.The screened monoclonal cells were injected into the peritoneal cavity of mice to prepare ascites.The monoclonal antibodies were identified by Western blotting detection,immunofluorescence assay(IFA)and antibody isotype analysis.【Result】The p30 protein prokaryotic expression plasmid was successfully constructed,and the recombinant p30 protein expressed in the prokaryotic system was purified using IPTG to induce.Four monoclonal antibody hybridoma cell lines,named 1H3B4,3F2F5,6E3A1 and 9D6B9,which could stably secrete the ASFV p30 protein,were successfully prepared.The potencies of the antibodies were determined by ELISA in the range of 1∶100000 to 1∶1000000.The screened monoclonal antibodies were identified by Western blotting and IFA and those antibodies were specifically reacted with p30 protein.The monoclonal antibodies were identified as IgG2b for the heavy chain and Kappa for the light chain of 3F2F5 and 9D6B9.6E3A1 had IgM for the heavy chain and Kappa for the light chain.1H3B4 had IgG2b for the heavy chain and Lambda for the light chain.【Conclusion】Four monoclonal antibodies against ASFV p30 protein were successfully prepared in this experiment,and the immunological characteristics of the monoclonal antibodies were analyzed,the results provided basis for the biological function research of p30 protein and the serological detection of ASFV.