肝癌相关的单克隆抗体的制备及其抗原表位分析

Prepared the monoclonal antibody associated with hepatocellular carcinoma and analyzed its epitope

目的:制备肝癌相关的单克隆抗体(mAb)用于肝癌的诊断。方法:用高转移肝癌细胞系HCCLM-6细胞免疫BALB/c小鼠后,取小鼠脾细胞与Sp2/0融合建立杂交瘤细胞系。通过ELISA法筛选HCCLM-6细胞蛋白的特异性mAb,用免疫组化染色法筛查对肝癌组织阳性率高的mAb,并用免疫荧光方法鉴定其在不同肿瘤细胞系中的表达,最后通过筛选Uni-ZAP XR人肝细胞cDNA表达文库初步鉴定mAb识别的抗原及其表位。结果:建立了28株杂交瘤细胞株,用ELISA法和免疫组化染色法筛选出肝癌阳性率较高的mAbQGA062。通过人肝细胞cDNA表达文库筛选初步鉴定表明mAb QGA062识别的抗原是纤维连接蛋白(FN),经分析其抗原表位位于肽段YTVSLVAIKGNQESPK。结论:获得与肝癌相关的mAb QGA062,并鉴定了其识别的抗原和表位,为肝癌的诊断和FN参与肝癌转移的机制研究提供了重要的制剂。

AIM：To prepare the monoclonal antibodies associated with hepatocellular carcinoma（HCC） for diagnosis.METHODS：3 BALB/c mice were immunized with high metastasis characteristic HCC cells（HCCLM-6）.The splenocytes from the immunized mice were fused with murine myeloma cells（Sp2/0）.The hybridoma cells were screened by ELISA after coating with the total protein and the culture supernatant of HCCLM-6 cells.The mAbs were characterized by immunohistochemical staining in the HCC tissues,and by indirect immunofIuorescent staining in different cell lines.The antigen and epitope recognized by the mAbs were identified by the screening premade Uni-ZAP human liver cDNA expression library.RESULTS：Twenty-eight hybridoma cells secreting mAbs were established.One clone of the hybridomas,QGA062,secreted specific mAb associated with HCC.The antigen recognized by the mAb QGA062 was identified as fibronectin（FN）,and the epitope was localized among the peptide YTVSLVAIKGNQESPK.CONCLUSION：The mAb against a HCC-associated epitope in FN is established and characterized,will be a very useful reagent for diagnosis of HCC.