人KIAA0350蛋白多克隆抗体制备及其应用

Preparation of polyclonal antibody of human KIAA0350 protein and its application

目的:表达、纯化带GST标签的人KIAA0350并制备多克隆抗体。方法:构建pGEX-4T-1-KIAA0350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 activatedSepharose TM4B进行纯化。用间接ELISA法检测抗体效价,同时用免疫组化检测细胞定位。结果:成功构建原核表达质粒,表达、纯化KIAA0350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∶4 000,应用该抗体检测肝组织中KIAA0350主要定位于细胞膜。结论:多克隆抗体的成功制备及其在肝组织中的表达定位,为进一步研究KIAA0350在糖尿病发病中的作用奠定了基础。

Objective：To express and purify human KIAA0350 with GST tag,prepare polyclonal antibody of human KIAA0350 protein.Methods：pGEX-4T-1-KIAA0350 prokaryotic expression plasmid was constructed and transformed into Esherichia coli BL21.The expression of fusion protein was induced by IPTG,after purification,the polyclonal antibody of human KIAA0350 protein was obtained by immunizing the Japanese white rabbits,and CNBr activated Sepharose TM4B was used for purification.Indirect ELISA was used to detect antibody titer,immunohistochemical method was used to detect the cellular localization.Results：The prokaryotic expression plasmid was constructed successfully,the polyclonal antibody of human KIAA0350 protein was obtained after expression and purification of KIAA0350 protein,and immunizing the animals.Indirect ELISA showed that the antibody titer was 1∶ 4 000.KIAA0350 was mainly located in cell membrane in liver tissue.Conclusion：The successful preparation of polyclonal antibody and its expression and localization in liver tissue further provide a basis for researching the effect of KIAA0350 in the pathogenesis of diabetes mellitus.