南方医科大学学报

南方医科大学学报 ›› 2015, Vol. 35 ›› Issue (12): 1715-.

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SA/hI-TAC双功能融合蛋白的制备及其生物学功能鉴定

许晓玲，柳英，陈青阁，黄通亮，高基民   

  • 出版日期:2015-12-20 发布日期:2015-12-20

Preparation and bioactivity evaluation of streptavidin-tagged human interferoninducible
T cell alpha chemoattractant bifunctional fusion protein

  • Online:2015-12-20 Published:2015-12-20

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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。

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.