人宫颈癌基因蛋白单克隆抗体的制备、鉴定及初步应用

Preparation, Characterization and Application of Anti-HCCR Monoclonal Antibody

目的:制备人宫颈癌基因蛋白单克隆抗体并初步用于肝癌血清检测。方法:重组表达HCCR-1蛋白胞外区(第167th-360th氨基酸)进行动物免疫,用含有HCCR蛋白抗原表位的融合蛋白筛选阳性克隆。通过ELISA、Western Blot、免疫组化鉴定抗体性质,并用于肝癌血清样本检测。结果:获得3株抗人HCCR蛋白单克隆抗体,其中B3抗体特异性、灵敏度较好。ELISA结果表明B3只识别GST-HCCRep蛋白和His-HCCR蛋白,与其他蛋白没有交叉反应;B3可检测到10 mg/L的GST-HCCRep蛋白;B3抗体亲和力常数Kaff=1.86×107(L/mol);Western Blot结果表明该抗体可识别天然HCCR蛋白;免疫组化检测表明肝癌组织中有HCCR蛋白表达而正常组织中未见表达;肝癌患者血清HCCR阳性率达到75%。结论:成功制备了HCCR单克隆抗体,为建立新的肝癌早期诊断方法奠定了基础。

Objective： To prepare monoclonal antibody （mAb） against HCCR. Methods： The extracellular segment of HCCR-1 （aa 167 to 360） was expressed and used to immunize Balb/c mice. Another protein GST-HCCRep, containing HCCR epitope （YLGTRR） was expressed and purified to detect the titers of serum antibody and screen the positive hybridomas. The mAb was characterized by ELI- SA, Western blot and immunohistochemistry. Results： The ELISA analysis indicated that the B3 mAb specifically recognized His-HCCR and GST-HCCRep protein, and showed no cross-reaction with other proteins; the detection limit of B3 mAb reached 10ng/ml of GST-HCCRep protein; the affinity constant of B3 mAb was 1.86× 10^7 L/tool; HCCR was found high-expressed in HCC cell lines HepG2 and 7721, as well as human HCC tissues. Neither normal hepatic cell line L02 nor normal liver tissues showed HCCR positive. The results showed that positive rate of HCCR in serum samples of HCC patients reached 58%. Conclusion： A specific mAb against HCCR was successfully generated and provided the foundation for future development of good methods for HCC diagnosis.