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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 and prepare polyclonal antibody. Methods: The prokaryotic expression vector pGEX-4T-1-KIAA0350 was constructed and transformed into E. coli BL21. The fusion protein was induced by IPTG. The purified polyclonal antibody was purified from the Japanese white rabbits and purified with CNBr activated Sepharose TM 4B. The antibody titer was detected by indirect ELISA and the cell localization was detected by immunohistochemistry. Results: The prokaryotic expression plasmid was successfully constructed, and the polyclonal antibody was obtained after the KIAA0350 protein was expressed and purified. Indirect ELISA showed that the antibody titer reached 1: 4000. Using this antibody to detect KIAA0350 in liver tissue, it mainly localized in the cell membrane. CONCLUSION: The successful preparation of polyclonal antibody and its expression in liver tissue lay the foundation for further study on the role of KIAA0350 in the pathogenesis of diabetes.