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目的:表达纯化ERCC1蛋白并制备其单克隆抗体(mAb)。方法:克隆并原核表达ERCC1蛋白,其氨基端带有6-His标签。纯化后的蛋白免疫BALB/c小鼠,经融合、筛选制备特异性mAb。结果:成功表达了ERCC1蛋白。SDS-PAGE显示所表达蛋白的相对分子质量(Mr)约为37000。获得了1株稳定分泌抗ERCC1抗体的杂交瘤细胞株(6F8),其分泌的mAb的Ig亚类(型)为IgG2b。ELISA检测,对应腹水mAb的效价为1∶1.5×106。Western blot结果显示抗ERCC1 mAb具有良好的特异性。结论:成功地表达纯化ERCC1蛋白并制备了1株抗ERCC1 mAb。
AIM: To express and purify ERCC1 protein and prepare its monoclonal antibody (mAb). Methods: The ERCC1 protein was cloned and expressed in prokaryotic cells with a 6-His tag at its amino terminus. BALB / c mice were immunized with the purified protein, and fused and screened to prepare specific mAb. Results: The ERCC1 protein was successfully expressed. The relative molecular mass (Mr) of the expressed protein was about 37,000 by SDS-PAGE. A hybridoma cell line stably secreting anti-ERCC1 antibody (6F8) was obtained, and the Ig subclass of the secreted mAb was IgG2b. ELISA, ascites mAb titers of 1: 1.5 × 106. Western blot results show that the anti-ERCC1 mAb has good specificity. Conclusion: ERCC1 protein was successfully expressed and purified and 1 anti-ERCC1 mAb was prepared.