Smarcad1重组蛋白表达及多克隆抗体验证

Expression and polyclonal antibody verification of Smarcad1 recombinant protein

目的:建立稳定的实验室多克隆抗体制备流程,选择Smarcad1作为目的基因进行重组蛋白表达纯化与多克隆抗体制备,并对其进行特异性验证。方法:以本实验室的pcDNA3.1-Flag-Smarcad1质粒作为模板,构建pET-16b-Smarcad1-F1原核表达质粒。表达Smarcad1-F1蛋白,并纯化。利用蛋白质免疫印迹,蛋白质免疫共沉淀和免疫荧光实验检测抗体特异性。结果:PCR鉴定结果显示,成功构建pET-16b-Smarcad1-F1表达质粒。蛋白表达纯化实验中,考马斯亮蓝染色检测,相较于诱导前,加入IPTG后Smarcad1蛋白成功诱导表达,且纯化出分子量为27 kD的目的蛋白。Western印迹证明,制备的抗体能够特异性识别内源性Smarcad1蛋白。蛋白质免疫共沉淀实验显示,相较于对照组,制备的抗体能够与Smarcad1蛋白结合。免疫荧光实验检测所制备抗体的特异性,结果显示制备的抗体可以特异性识别内源性Smarcad1蛋白。结论:成功构建了Smarcad1原核表达质粒,利用原核蛋白表达纯化出Smarcad1蛋白,利用该蛋白制备的抗体可以特异性识别内源性Smarcad1蛋白,为后续原核表达蛋白及多克隆抗体制备提供思路。

Objective:To establish a stable laboratory polyclonal antibody preparation process,to select Smarcad1 as the target gene for recombinant protein expression purification and polyclonal antibody preparation,and to verify its specificity.Methods:The pcDNA3.1-Flag-Smarcad1 plasmid in our laboratory was used as the template to construct the pET-16b-Smarcad1-F1 prokaryotic expression plasmid.Smarcad1-F1 protein was expressed and purified.Antibody specificity was detected using Western blotting,protein co-immunoprecipitation and immunofluorescence experiments.Results:PCR identification results showed that the pET-16b-Smarcad1-F1 expression plasmid was successfully constructed.In the protein expression purification experiment,Coomassie brilliant blue staining showed that compared with before induction,the Smarcad1 protein was successfully induced to express after adding IPTG,and the target protein with a molecular weight of 27 kD was purified.Western blotting proved that the prepared antibody could specifically recognize the endogenous Smarcad1 protein.The protein co-immunoprecipitation experiment showed that,compared with the control group,the prepared antibody could bind to the Smarcad1 protein.The specificity of the prepared antibody was detected by immunofluorescence experiments,and the results showed that the prepared antibody could specifically recognize the endogenous Smarcad1 protein.Conclusion:The Smarcad1 prokaryotic expression plasmid is successfully constructed,and the Smarcad1 protein is purified by prokaryotic protein expression.The antibody prepared from this protein can specifically recognize the endogenous Smarcad1 protein,which provides ideas for the subsequent prokaryotic expression protein and polyclonal antibody preparation.