猴痘病毒特异性单克隆抗体制备及鉴定

Generation and characterization of specific monoclonal antibodies against monkeypox virus

目的 制备猴痘病毒特异性单克隆抗体,建立猴痘病毒免疫荧光检测方法.方法 原核表达猴痘病毒A29蛋白及与之同源的痘苗病毒A27蛋白和牛痘病毒162蛋白,建立抗体筛选系统;使用人工合成的猴痘病毒A2917~49 多肽免疫BALB/c小鼠,取小鼠脾细胞通过融合、克隆和筛选等制备猴痘病毒单克隆抗体.通过ELISA法确定单克隆抗体的特异性及亚型.结果 在大肠埃希菌中成功表达A29、A27及162蛋白,经Western blot鉴定分子量与预期相符,带his标签的三种蛋白经His-Bind亲和层析柱纯化后纯度均大于90%;用纯化蛋白筛选获得8株A29蛋白特异的单克隆抗体,其中两株为IgG3型,其余为IgG1型.结论 本研究获得8株猴痘病毒特异性单克隆抗体,为进一步建立猴痘病毒免疫荧光检测奠定基础.

Objective To generate monkeypox virus specific monoclonal antibodies for further establishing monkeypox virus immunofluorescence assay.Methods Monkeypox virus A29 protein, vaccinia ortholog A27 protein and cowpox ortholog 162 protein were expressed in E.coli BL21 to screen antibodies.Synthetic monkeypox virus A2917 ~ 49 polypeptide was used to immune BALB/c mice.Monkeypox virus monoclonal antibodies were generated through fusion, cloning and screening techniques.Indirect ELISA was performed to test antibodies specificity and subtype.Results A29, A27 and 162 proteins were highly expressed in E.coli and detected by Western blot.The three his-tagged proteins were purified using His-Bind affinity chromatography column.The purity of the proteins was all more than 90%.And 8 strains monkeypox virus specific monoclonal antibodies were screened by the three purified proteins.Two mAbs of 8 were IgG3 subtype and the rest were IgG1 subtype.Conclusions Eight strains of monkeypox virus specific monoclonal antibodies were generated, they can be used to further establish monkeypox virus immune immunofluorescence assay.