人DFF45蛋白多克隆抗体制备及其初步应用

Preparation and Preliminary Application of Human Protein DFF45 Polyclonal Antibody

目的:表达、纯化带GST标签的DNA裂解因子45(DNA fragmentation factor 45,DFF45)融合蛋白并制备多克隆抗体。方法:构建pGEX-5X-1/DFF45原核表达质粒,转化大肠杆菌BL21,用IPTG诱导融合蛋白表达,经纯化后免疫日本大耳白兔得到多克隆抗体并用CNBr-activatedSepharoseTM4B进行纯化。用间接ELISA法检测抗体效价,Western blot鉴定抗体特异性,同时用免疫荧光染色鉴定抗体特异性并观察DFF45的细胞定位。进一步检测DFF45在人类几种细胞系的表达差异情况。结果:成功构建原核表达质粒,表达、纯化DFF45蛋白并免疫动物后得到多克隆抗体。间接ELISA法显示抗体效价达1∶20 000,Western blot确定抗体具有高度特异性。应用该抗体检测发现DFF45在人类几种细胞系中表达存在差异并观察到其细胞定位。结论:DFF45多克隆抗体的成功制备及其在人类细胞系的差异表达,为进一步研究DFF45基因与肿瘤及其相关疾病的关系奠定了基础。

To express and purify the fusion protein of GST-tagged DNA fragmentation factor 45（DFF45） in prokaryocytes and prepare anti-DFF45 polyclonal antibody.The expression plasmid pGEX-5X-1/DFF45 was constructed and transformed into E.coli BL21,and the expression of GST-DFF45 protein was induced by IPTG.After purification,the fusion protein GST-DFF45 was used to immunize Oryctolagus cuniculus to obtain the antibody serum.Then the anti-DFF45 serum was purified by CNBr-activated SepharoseTM 4B.The titer of the polyclonal antibody was detected by ELISA and its specificity was analyzed by Western blotting and immunofluorescence.Furthermore,the expression of DFF45 protein in several human cell lines was detected,and the cellular localization of DFF45 protein was observed.The fusion protein was successfully expressed and purified,and the specific polyclonal antibody was prepared.The titer of the polyclonal antibody was up to 1：20 000 detected by ELISA,and the high specificity of the antibody was confirmed by Western blotting and immunofluorescence.Moreover,it is found that DFF45 protein showed differential expression in several human cell lines;and that DFF45 protein was located in both cytoplasm and nucleus.The successful preparation of DFF45 polyclonal antibody and the confirmation of its different expression levels in human cells provides a tool for the further study of the relation between DFF45 and tumors.