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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 human B lymphocyte stimulating factor - diphtheria toxin fusion protein (hsBAFF-DT388) and investigate its target B cell activity. Methods According to the optimized hsBAFF-DT388 gene sequence, primers were designed. The target gene was amplified by PCR and inserted into the cloning vector pMD19-T. The positive clones were identified by colony PCR, enzyme-linked immunosorbent assay and DNA sequencing. The recombinant cloned vector was double digested and inserted Vector pCold Ⅱ, and identify recombinant expression vector. The recombinant strain was induced by ITPG, identified by SDS-PAGE and Western blot. The recombinant protein was purified by Ni2 + -NTA column and the biological activity of the recombinant protein was tested. Results hsBAFF-DT388 high expression strains, the target protein accounted for about 40% of the total bacterial protein. The recombinant protein specifically binds to BAFF receptor positive cells and has a targeted cytotoxic effect on the human B cell line Hmy2.CIR. Conclusion The hsBAFF-DT388 expression vector was successfully constructed to obtain a recombinant protein with targeted B cell activity, which laid the foundation for its application in the treatment of B-cell malignancies and autoimmune diseases.