人DFF45蛋白多克隆抗体制备及其初步应用

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-activated Sepharose? 4B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。

Preparation and Preliminary Application of Human Protein DFF45 Polyclonal Antibody

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 polyc...