摘要
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：To clone,express human Golgi protein 73（GP73）,develop and characterize the monoclonal antibody（mAb） against GP73,a valuable serum marker for hepatocellular carcinoma（HCC）.Methods： GP73 gene was amplified by PCR,and cloned into prokaryotic expression vector pComb3XSS.The recombinant construct was expressed in Top10F′ E.coli.host,and purified with their fusion partner by HisFF Trap affinity chromatography.The recombinant protein was used to immunize the BALB/c mice for mAb development.The titer of the mAb was detected by ELISA and its specificity was analyzed by Western blot.The sera level of GP73 in HCC patients and healthy people was measured by co-immunoprecipitation（IP）.Results： The GP73-6×His recombinant protein was successfully expressed and purified.Five hybridoma cell lines against GP73 were obtained.IP revealed that the mAb could combine with GP73 in human sera with high specificity.The level of GP73 in HCC patients is much higher than that of healthy people.Conclusion： The success in mouse a-GP73 mAb development provides the basis for further developing a sandwich ELISA for detection of human GP73.
出处
《南京医科大学学报（自然科学版）》
CAS
CSCD
北大核心
2012年第3期301-305,共5页
Journal of Nanjing Medical University(Natural Sciences)
基金
江苏省卫生厅科研项目(H200814)
江苏省卫生厅医学重点人才课题(RC2011082)
