SA/hI-TAC双功能融合蛋白的制备及其生物学功能鉴定  

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

作　　者：许晓玲[1] 柳英 陈青阁[1] 黄通亮 高基民[1] 

机构地区：[1]温州医科大学//浙江省模式生物技术与应用重点实验室,浙江温州325035 [2]烟台龙矿中心医院,山东烟台265700

出　　处：《南方医科大学学报》2015年第12期1715-1720,共6页Journal of Southern Medical University

基　　金：国家863计划重大项目(2012AA02A407)~~

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/h I-TAC),并对其生物学功能进行鉴定。方法构建p ET24a-SA-h I-TAC/p ET21a-h I-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h I-TAC-SA的趋化作用明显强于SA-h I-TAC。结论 SA/h I-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。Objective To prepare streptavidin- tagged human interferon- inducible T cell alpha chemoattractant bifunctional fusion proteins（SA/h I- TAC） and evaluate its biological activity. Methods p ET24a- SA- h I- TAC/p ET21a- h I- TAC- SA plasmids were constructed and expressed in BL21. SA- h I- TAC and h I- 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（biotinbinding function and h I-TAC activity） was analyzed by flow cytometry and lymphocyte chemotaxis experiment, respectively.Results SA- h I- TAC/h I- 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- h I- TAC and 98.8% for h I- TAC- SA）. SA/h I- TAC induced lymphocyte chemotaxis in a dose- dependent manner, and h I- TAC- SA showed a stronger chemotactic effect than SA- h I- TAC.Conclusion We successfully obtained SA/h I- TAC bifunctional fusion proteins, which may potentially be used in local treatment of tumor and as a tumor vaccine.

关 键 词：人干扰素诱导T细胞α趋化因子 链亲和素 融合蛋白 表面修饰 

分 类 号：R730.5[医药卫生—肿瘤]