人sBAFF-DT388融合蛋白在大肠杆菌中的表达及其活性研究

目的 制备人B淋巴细胞刺激因子-白喉毒素融合蛋白(hsBAFF DT388)，探讨其靶向B细胞活性。方法 根据优化合成的hsBAFF DT388基因序列设计引物，PCR扩增目的基因并插入克隆载体pMD19-T，采用菌落PCR、酶谱分析及DNA测序鉴定阳性克隆，重组克隆载体经双酶切后插入表达载体pColdⅡ，并鉴定重组表达载体。重组菌株经ITPG诱导，SDS-PAGE分析及Western blot鉴定，经Ni2+ NTA层析柱纯化，检测重组蛋白的生物学活性。结果 获得hsBAFF-DT388高效表达菌株，目的蛋白占菌体总蛋白的40%左右。重组蛋白与BAFF受体阳性细胞特异性结合并对人B细胞系Hmy2.CIR具有靶向细胞毒作用。结论 成功构建hsBAFF DT388表达载体，获得具有靶向B细胞活性的重组蛋白，为其在B细胞恶性肿瘤及自身免疫性疾病治疗中的应用研究奠定基础。

Expression of the fusion protein of human soluble B lymphocyte stimulator and diphtheria toxin in E.coli and its biological activity

Objective To prepare the fusion protein of human soluble B lymphocyte stimulator and diphtheria toxin(hsBAFF-DT388) in E.coli and investigate its targeted activity to human B cells.Methods The hsBAFF-DT388 gene was optimized in advance and inserted into the clone vector pMD19-T.The fragment of the hsBAFF-DT388 fusion gene was separated from the plasmid pUC57-hsBAFF-DT388 by PCR according to hsBAFF-DT388 gene order.The recombinant was screened with colony PCR,restriction map analysis and DNA sequencing.The r...