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目的:高尔基体蛋白73(Golgi protein 73,GP73)是新近发现的肝细胞性肝癌(hepatocellular carcinoma,HCC)的重要血清学标志物。通过克隆、表达人GP73,制备抗GP73单克隆抗体并鉴定其特性。方法:克隆GP73基因,构建GP73原核表达载体pComb3XSS-GP73,诱导表达,以HisFF Trap亲和层析柱纯化GP73-6×His重组融合蛋白。以该融合蛋白免疫BALB/c小鼠,制备抗GP73单克隆抗体。以间接法ELISA检测抗体效价;Western blot鉴定抗体特异性;免疫共沉淀法初步检测肝癌患者血清中GP73表达水平。结果:成功表达并纯化了GP73-6×His重组蛋白;获得5株稳定分泌抗人GP73单克隆抗体杂交瘤细胞株;免疫共沉淀证实抗GP73单克隆抗体能与肝癌患者血清中GP73蛋白特异性结合,GP73水平在肝癌患者血清中较正常人显著升高。结论:成功制备了抗人GP73单克隆抗体,为后期建立检测人GP73的双抗体夹心ELISA方法打下基础。
OBJECTIVE: Golgi protein 73 (GP73) is an important serological marker of newly discovered hepatocellular carcinoma (HCC). By cloning and expressing human GP73, an anti-GP73 monoclonal antibody was prepared and characterized. Methods: The GP73 gene was cloned and the prokaryotic expression vector pComb3XSS-GP73 was constructed. The GP73-6 × His recombinant fusion protein was purified by HisFF Trap affinity chromatography. BALB / c mice were immunized with the fusion protein to prepare anti-GP73 monoclonal antibodies. The antibody titer was detected by indirect ELISA. The specificity of antibody was detected by Western blot. The expression of GP73 in serum of hepatocellular carcinoma patients was detected by co-immunoprecipitation. RESULTS: GP73-6 × His recombinant protein was successfully expressed and purified. Five strains of monoclonal antibodies against human GP73 were successfully secreted. Co-immunoprecipitation demonstrated that the anti-GP73 monoclonal antibody could specifically antagonize the GP73 protein in serum of hepatocellular carcinoma patients Combined, GP73 levels in serum of patients with liver cancer significantly higher than normal. CONCLUSION: Monoclonal antibody against human GP73 was successfully prepared and laid the foundation for the establishment of a sandwich ELISA method for the detection of human GP73 in the later stage.