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目的:制备抗人乳腺癌候选抑制蛋白1(BCSC-1)蛋白单克隆抗体(mAb),并对其特异性进行鉴定。方法:构建原核表达载体pET-30a-BCSC-1,在原核表达系统E.coliBL21(DE3)中表达重组人BCSC-1蛋白。超声洗涤纯化重组蛋白作为抗原免疫BALB/c小鼠,取免疫小鼠的脾细胞和同系小鼠的骨髓瘤细胞Sp2/0进行常规融合,通过有限稀释法进行克隆和间接ELISA筛选,获得分泌小鼠抗人BCSC-1蛋白mAb的杂交瘤细胞株,通过ELISA、Western blot等方法检测其特异性。将真核重组表达载体pcDNA3.1/v5-HisB-BCSC-1转染入乳腺癌MCF-7细胞,通过免疫组化确定了人BCSC-1蛋白的表达。结果:成功构建了原核表达载体pET-30a-BCSC-1,经IPTG诱导表达出了重组人BCSC-1蛋白,主要以包涵体的形式存在沉淀中。用纯化的重组BCSC-1蛋白免疫小鼠后经融合筛选得到1株稳定分泌抗BCSC-1的mAb杂交瘤细胞株。通过ELISA、Western blot、免疫组化等方法鉴定,抗BC-SC-1的mAb能与BCSC-1蛋白特异性结合。结论:成功制备了小鼠抗人BCSC-1的mAb。
Objective: To prepare monoclonal antibody against human breast cancer candidate inhibitor 1 (BCSC-1) and identify its specificity. Methods: The prokaryotic expression vector pET-30a-BCSC-1 was constructed and the recombinant human BCSC-1 protein was expressed in prokaryotic expression system E.coli BL21 (DE3). The recombinant protein was purified by ultrasonic washing and then used as antigen to immunize BALB / c mice. Sp2 / 0 of spleen cells of immunized mice and myeloid cells of normal mice were routinely fused and cloned by indirect dilution ELISA. The anti-human BCSC-1 protein mAb hybridoma cell lines were detected by ELISA, Western blot and other methods to detect its specificity. The eukaryotic recombinant expression vector pcDNA3.1 / v5-HisB-BCSC-1 was transfected into breast cancer MCF-7 cells, and the expression of human BCSC-1 protein was confirmed by immunohistochemistry. Results: The prokaryotic expression vector pET-30a-BCSC-1 was successfully constructed. Recombinant human BCSC-1 protein was induced by IPTG, and mainly existed in the form of inclusion bodies. After the mice were immunized with the purified recombinant BCSC-1 protein, one strain of monoclonal antibody secreting anti-BCSC-1 mAb was obtained by fusion screening. The anti-BC-SC-1 mAbs could specifically bind to BCSC-1 protein by ELISA, Western blot and immunohistochemistry. Conclusion: Mouse anti-human BCSC-1 mAb was successfully prepared.