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目的构建重组hMAM蛋白,通过杂交瘤技术获得特异性的抗hMAM的单克隆抗体,并鉴定其生物特性。方法克隆表达hMAM 8-93AA、20-52AA、43-70AA、56-82AA、67-93AA区段的抗原,以纯化的pBV220/hMAM(8-93AA)为免疫原,杂交瘤技术筛选出能稳定分泌抗hMAM单克隆抗体的细胞株。采用Western blotting和免疫组织化学染色方法鉴定其表位和特异性。结果获得6个高纯度的重组抗原,通过pBV220/hMAM(8-93AA)免疫小鼠后,获得2株稳定分泌抗hMAM抗体的杂交瘤细胞株。Western blotting结果显示MAb1、MAb2分别是识别hMAM20-52AA、hMAM52-67AA片段的特异性单克隆抗体。免疫组织化学染色结果显示该抗体仅在乳腺组织中呈阳性反应,在其他肿瘤组织中不反应。结论通过杂交瘤技术成功制备了两株稳定分泌且高特异性的抗hMAM不同位点的单克隆抗体细胞株,为进一步研制高灵敏的乳腺癌早期检测试剂奠定了基础。
Objective To construct recombinant hMAM protein, obtain specific anti-hMAM monoclonal antibody by hybridoma technique and identify its biological characteristics. Methods The antigens of hMAM 8-93AA, 20-52AA, 43-70AA, 56-82AA and 67-93AA were cloned and purified with the purified pBV220 / hMAM (8-93AA) Cell lines secreting anti-hMAM monoclonal antibodies. Western blotting and immunohistochemistry were used to identify the epitope and specificity. Results Six high purity recombinant antigens were obtained. After the mice were immunized with pBV220 / hMAM (8-93AA), two hybridoma cell lines stably secreting anti-hMAM antibody were obtained. Western blotting showed that MAb1 and MAb2 were specific monoclonal antibodies that recognize hMAM20-52AA and hMAM52-67AA, respectively. Immunohistochemical staining results showed that the antibody was positive only in breast tissue, not in other tumor tissues. Conclusion Two monoclonal antibody cell lines stably secreting and highly specific against different sites of hMAM were successfully prepared by hybridoma technique, which laid the foundation for the further development of highly sensitive detection reagents for breast cancer.