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目的制备高纯度的B细胞活化因子(BAFF)可溶性突变体(smBAFF)蛋白,鉴定其生物学活性。方法重组原核表达载体pET41a/smBAFF在大肠杆菌BL21中经IPTG诱导表达smBAFF蛋白,采用SDS-PAGE和Western blot检测表达产物,超声碎菌,提取包涵体,Ni2+-NTA亲和层析纯化,复性,鉴定其生物学活性。结果经鉴定表达出相对分子质量为1.7×104的外源蛋白smBAFF,经Ni2+-NTA亲和层析纯化出该重组蛋白,复性后的smBAFF与B细胞具有较高的亲和力,但失去共刺激B细胞增殖的能力,且能竞争性抑制天然sBAFF的作用。结论成功制备具有B细胞结合活性而失去刺激B细胞增殖活性的smBAFF,为以smBAFF为靶向载体在B细胞恶性增殖性和异常活化性疾病治疗研究奠定基础。
Objective To prepare high purity BAFF soluble mutant (smBAFF) protein and identify its biological activity. Methods Recombinant prokaryotic expression vector pET41a / smBAFF was expressed in Escherichia coli BL21 induced by IPTG. The expressed product was detected by SDS-PAGE and Western blot. The inclusion bodies were extracted and purified by Ni2 + -NTA affinity chromatography. , Identification of its biological activity. Results The expressed foreign protein smBAFF with a molecular weight of 1.7 × 104 was identified and purified. The recombinant protein was purified by Ni2 + -NTA affinity chromatography. The refolded SMBAFF had higher affinity with B cells, but lost costimulation B cell proliferation, and competitive inhibition of the role of native sBAFF. Conclusion The successful preparation of smBAFF with B-cell binding activity and loss of stimulatory B-cell proliferative activity has laid the foundation for the study of treatment of malignant proliferative and abnormal activation of B-cell with smBAFF as a targeted vector.