人sBAFF-DT388融合蛋白在大肠杆菌中的表达及其活性研究

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

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。

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 recombinant vector was digested by restriction enzymes,and then inserted into the expression vector pColdⅡ.The positive recombinant expression vector was identified by colony PCR and restriction map analysis.The recombinant strain was induced by IPTG.The recombinant protein was identified by SDS-PAGE and Western blot,and then purified by Ni2＋-NTA affinity chromatography.Biological activity of the purified protein was detected by cell fluoresce and MTT assay.Results Expression level of the recombinant protein accounted for 40% of total bacterial proteins of E.coli,and the recombinant protein could bind with BAFF receptor-positive cells and had a cytotoxic effect on human B cells（Hmy2.CIR）.Conclusion The fusion protein expression vector of hsBAFF-DT388 was successfully constructed and the recombinant protein with selective cytotoxicity against B cell was obtained,which may establish a solid foundation for further study of the therapy for B cell malignancies and autoimmune diseases.