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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。
Objective: To prepare hepatoma-associated monoclonal antibody (mAb) for the diagnosis of liver cancer. Methods: BALB / c mice were immunized with HCCLM-6 cells with high metastatic potential. The spleen cells were fused with Sp2 / 0 to establish a hybridoma cell line. Specific mAb of HCCLM-6 cells was screened by ELISA, mAb positive to HCC was screened by immunohistochemistry, and its expression in different tumor cell lines was identified by immunofluorescence. Finally, ZAP XR human hepatocyte cDNA expression library Preliminary identification of antigens recognized by mAbs and their epitopes. Results: 28 hybridoma cell lines were established. The mAb QGA062 with the highest positive rate of HCC was screened by ELISA and immunohistochemistry. Preliminary identification of human liver cell cDNA expression library screening showed that the antigen recognized by mAb QGA062 was fibronectin (FN), and its antigenic epitope was located on the peptide YTVSLVAIKGNQESPK. Conclusion: Obtaining the mAb QGA062 associated with hepatocellular carcinoma and identifying its identified antigens and epitopes provide important preparations for the diagnosis of liver cancer and the mechanism of FN involved in the metastasis of liver cancer.