人KIAA1199蛋白表达、纯化、抗体制备及鉴定

Expression,purification,antibody preparation and identification of KIAA1199

目的 KIAA1199是一种功能未知的胞浆蛋白,通过制备多克隆抗体来为进一步研究其功能奠定基础。方法采用PCR技术扩增出目的基因,将其克隆到pMAL-C2X和pGEX-5X-1原核表达载体中。经IPTG诱导后,重组质粒pMAL-C2X/KIAA1199和pGEX-5X-1/KIAA1199,分别表达含麦芽糖结合蛋白(MBP)的MBP-KIAA1199融合蛋白和含重组谷胱甘肽转移酶(GST)的融合蛋白GST-KIAA1199。其中,MBP-KIAA1199融合蛋白经纯化后,免疫新西兰大白兔,制备多克隆抗体;GST-KIAA1199融合蛋白用于检测抗体特异性。此后应用ELISA及Westernblot方法鉴定该抗体。结果 pMAL-C2X/KIAA1199和pGEX-5X-1/KI-AA1199两个重组质粒构建成功,并获得了高效价的特异性多克隆抗体。结论成功制备了KIAA1199抗体,为进一步研究基因功能奠定了基础。

Objective To prepare the polyclonal antibody of an unknown cytosolic protein KIAA1199 for the following function study.Methods Target genes were amplified by PCR and cloned into prokaryotic expression vector pMAL-C2X and pGEX-5X-1,so the recombinant prokaryotic expression vector pMAL-C2X/KIAA1199 and pGEX-5X-1/KIAA1199 were constructed,and the corresponding fusion proteins maltose binding protein MBP-KIAA1199 and GST-KIAA1199 were induced with IPTG separately.After purification,MBP-KIAA1199 fusion protein was used to immunize New Zealand rabbits to prepare polyclonal antibody,and GST-KIAA1199 was used to identify the specificity of the new antibody.Thereafter,the antibody was detected by Western blot and ELISA.Results Two recombinant prokaryotic expression vectors were constructed successfully,and specific polyclonal antibodies were also generated.Conclusions Preparation of KIAA1199 polyclonal antibody successfully,which may lay foundation to investigating the function of KIAA1199 gene in the future.