抗扎伊尔型埃博拉病毒单克隆抗体的制备和鉴定

Preparation and identification of monoclonal antibody against Zaire Ebola virus

目的采用扎伊尔型埃博拉病毒核蛋白(EBOV NP)的合成多肽为免疫原制备鼠源单克隆抗体,并用4种埃博拉流行株核蛋白基因的真核表达蛋白对制备的单克隆抗体进行特异性分析。方法用人工合成的扎伊尔型埃博拉病毒核蛋白多肽免疫动物,进行细胞融合后筛选阳性杂交瘤细胞。构建埃博拉病毒4种亚型的真核表达载体转染HEK293T细胞以表达目的蛋白,再以真核表达蛋白为抗原,用免疫印迹方法分析扎伊尔型埃博拉病毒单克隆抗体的特异性。结果完成了抗扎伊尔型单克隆抗体的制备,共得到能分泌抗扎伊尔型埃博拉病毒单克隆抗体的杂交瘤细胞12株,小鼠腹水抗体效价介于1∶104~1∶105,其中3株杂交瘤细胞株分泌的抗扎伊尔型埃博拉病毒单克隆抗体与其他3种亚型无交叉反应,抗体效价高、特异性好。结论成功制备抗扎伊尔型埃博拉病毒单克隆抗体,为建立快速检测埃博拉病毒的方法奠定基础。

Zaire Ebola virus nucleoprotein （EBOV NP） synthetic peptide was used as the immunogen to prepare mouse monoclonal antibody, and the specificity of prepared monoclonal antibody was tested with recombinant nucleoproteins of four Ebola virus epidemic strains.The artificially synthesized nucleoprotein peptides of Zaire Ebola virus were used to immunize animals, and hybridoma cell lines were screened after the cells fusion.Four eukaryotic expression vectors of the Ebola virus subtype were constructed to transfect HEK293T cell to express target proteins. Then the eukaryotic expression proteins were used as antigens to test the specificity of prepared monoclonal antibody against Zaire EBOV by Western blotting. We accomplished the preparation of monoclonal antibody against Zaire EBOV, and twelve cell lines that can secret monoclonal antibodies against Zaire EBOV were obtained,the titers of antibodies in mouse ascites were among 1∶104-1∶105. Three hybridoma cell lines of the twelve were screened, which secret antibodies that have no cross reaction with other three Ebola virus subtype, and these antibodies had high titer and specificity. In conclusion, the monoclonal antibody against Zaire Ebola virus was successfully prepared, which laid the foundation for the rapid detection of Ebola virus.