SA/hI-TAC双功能融合蛋白的制备及其生物学功能鉴定 |
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引用本文: | 许晓玲,柳英,陈青阁,黄通亮,高基民.SA/hI-TAC双功能融合蛋白的制备及其生物学功能鉴定[J].南方医科大学学报,2015,35(12):1715. |
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作者姓名: | 许晓玲 柳英 陈青阁 黄通亮 高基民 |
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摘 要: | 目的制备链亲和素连接的人干扰素诱导T细胞α趋化因子融合蛋白(SA/hI-TAC),并对其生物学功能进行鉴定。方法构 建pET24a-SA-hI-TAC/pET21a-hI-TAC- SA表达载体,在大肠杆菌BL21中诱导表达两种融合蛋白,用镍金属螯合层析纯化、透 析复性及蛋白质印迹法(Western blot)鉴定,融合蛋白中I-TAC部分的生物学活性由淋巴细胞趋化实验检测;融合蛋白中SA的 生物学活性由流式细胞仪测定。结果两种融合蛋白可在大肠杆菌BL21 中被诱导表达,分别占细菌表达总蛋白量的12%和 25%,经镍柱纯化后融合蛋白纯度达85%、90%,经丙烯葡聚糖凝胶S-100过滤层析后,纯度均可达到98%,融合蛋白在生物素化 的MB49细胞(小鼠膀胱癌细胞)表面的修饰效率分别为91.3%、98.8%,并对淋巴细胞的趋化作用呈剂量依赖性,且hI-TAC-SA 的趋化作用明显强于SA-hI-TAC。结论SA/hI-TAC 双功能融合蛋白可能应用于肿瘤局部治疗以及肿瘤疫苗。
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Preparation and bioactivity evaluation of streptavidin-tagged human interferoninducibleT cell alpha chemoattractant bifunctional fusion protein |
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Abstract: | Objective To prepare streptavidin-tagged human interferon-inducible T cell alpha chemoattractant bifunctional fusion proteins (SA/hI-TAC) and evaluate its biological activity. Methods pET24a-SA-hI-TAC/pET21a-hI-TAC-SA plasmids were constructed and expressed in BL21. SA-hI-TAC and hI-TAC-SA fusion proteins were purified by Ni-NTA affinity chromatography, refolded by dialysis and identified by Western blotting. The bifunctionality of the fusion proteins (biotin-binding function and hI-TAC activity) was analyzed by flow cytometry and lymphocyte chemotaxis experiment, respectively. Results SA-hI-TAC/hI-TAC-SA fusion proteins were expressed at about 12% and 25% of the total bacterial protein, respectively. The two fusion proteins had a purity of about 85% and 90% after purification, and their purity reached 98% after purification with S-100 gel filtration chromatography. Both of the fusion proteins were efficiently immobilized on the surface of biotinylated mouse bladder cancer MB49 cells (91.3% for SA-hI-TAC and 98.8% for hI-TAC-SA). SA/hI-TAC induced lymphocyte chemotaxis in a dose-dependent manner, and hI-TAC-SA showed a stronger chemotactic effect than SA-hI-TAC. Conclusion We successfully obtained SA/hI-TAC bifunctional fusion proteins, which may potentially be used in local treatment of tumor and as a tumor vaccine.
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