小鼠Rnf141多克隆抗体的制备及鉴定

Polyclonal Antibody Preparation and Characterization of Mouse Rnf141

目的获得小鼠Rnf141多克隆抗体,并对其功能进行初步研究。方法利用多聚酶链反应(PCR)扩增获得鼠Rnf141基因并且与原核表达载体pGEX-5X-3(含GST标签)进行重组,将重组质粒转入大肠杆菌BL21(DE3),经1mmol/L IPTG在25℃条件下诱导,用SDS-PAGE检测,获得GST-Rnf141融合蛋白高效表达后,通过Glutathione Sephorase4B纯化。纯化的融合蛋白免疫家兔制备多克隆抗体,通过Western blot和免疫组织化学的方法检测抗体的特异性。结果成功构建了重组质粒pGEX-Rnf141,该质粒能在大肠杆菌BL21(DE3)中表达融合蛋白GST-Rnf141,融合蛋白表达量约占细菌总蛋白的46%,制备的多抗效价达1∶128000,它可以与GST-Rnf141重组蛋白和小鼠不同脏器组织Rnf141蛋白发生特异的抗原抗体反应,在脾脏和脑组织中Rnf141蛋白表达量较高。结论成功制备小鼠Rnf141多克隆抗体,该抗体为研究Rnf141的表达与功能提供了有用的工具。

Objective To obtain the Rnf141 polyclonal antibody and to study its function. Methods Rnf141 cDNA was obtained by PCR amplification and inserted into the expression vector pGEX-5X-3, which encoding the GST protein to generate a recombinant plasmid. The recombinant was transformed into E. coli BL21 （DE3）. After inducing with 1 mmol/L IPTG at 25 ℃, the GST-Rnf141 fusion protein was expressed at a high level as a soluble form. Purified fusion protein with Glutathione Sephorase 4B chromatography was applied to immune rabbits to prepare the polyclonal, antibody. Western blot and immunobistoehemistry were applied to confirm the specificity of the resulting antibody. Results pGEX-Rnf141 recombinant plasmid was constructed successfully and the resulting. GST- Rnf141 fusion protein could be expressed in E. coli BL21 （DE3） at a relatively high level, standing for 46% of total bacteria protein. The titer of the antiserum was 1 ： 128000 and the polyclonal antibody could recognize GST- Rnf141 fusion protein and Rnf141 protein in different tissues of mouse. The high expression of Rnf141 protein was observed in spleen and brain of mouse. Conclusion Rnf141 polyclonal antibody was prepared successfully and this will provide material for further studies of Rnf141 expression and function.