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目的:制备并鉴定抗GST、His×6及FLAG的单克隆抗体(mAb)。方法:分别以含GST、His×6及FLAG标签的融合蛋白为抗原免疫BALB/c小鼠,通过细胞融合制备抗相应标签蛋白的mAb。用Westernblot检测mAb对变性融合蛋白的反应性。用流式细胞术(FCM)检测抗FLAGmAb对细胞膜表面融合蛋白的反应性。结果:共获得12株可稳定分泌抗3种标签蛋白mAb的杂交瘤细胞(其中5株可分泌抗GSTmAb,5株可分泌抗His×6mAb,2株可分泌抗FLAGmAb)。11株mAb均可用于相应融合蛋白的Westernblot检测。1株抗FLAGmAb检测细胞膜表面融合蛋白的阳性率,与商品化的mAbM2相接近。用1株抗His×6mAb制备亲和层析柱,并用于纯化IL-8-His融合蛋白,也获得较满意的结果。结论:成功地制备了抗GST、抗His×6及抗FLAG的mAb,为含标签的融合蛋白的研究和应用提供了重要的工具。
Objective: To prepare and identify monoclonal antibodies (mAbs) against GST, His × 6 and FLAG. Methods: BALB / c mice were immunized with fusion protein containing GST, His × 6 and FLAG respectively, and the mAbs against the corresponding tag protein were prepared by cell fusion. Reactivity of mAbs to denatured fusion proteins was detected by Western blot. Flow cytometry (FCM) was used to test the reactivity of anti-FLAG mAbs to cell membrane surface fusion proteins. Results: Twelve hybridomas secreting anti-GST mAb, 5 secretable anti-His × 6 mAb and 2 anti-FLAG mAb secreting anti-3 tag mAb were obtained. All 11 mAbs could be used for Western blot analysis of the corresponding fusion proteins. One anti-FLAG mAb detected the positive rate of cell surface fusion protein, which was close to that of commercial mAbM2. An anti-His × 6 mAb was used to prepare the affinity chromatography and to purify the IL-8-His fusion protein. Satisfactory results were also obtained. Conclusion: The anti-GST, anti-His × 6 and anti-FLAG mAbs were successfully prepared and provided an important tool for the research and application of tagged fusion proteins.