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目的获得人类B细胞激活因子(B cell activating factor,BAFF)胞外段融合蛋白并对其生物活性进行分析。方法构建pET32a/BAFF的原核表达载体,进行原核表达、亲和纯化,并通过SDS-PAGE、Western印迹进行鉴定。利用非还原电泳后的Western印迹与HPLC分析融合蛋白在溶液中的存在形式。ELISA、Octet RED系统检测rhBAFF结合受体TACI的能力并做出实时动力学分析。MTS法检测rhBAFF促进B细胞增殖的活性。结果融合蛋白在大肠杆菌BL21中以可溶形式高效表达,经Ni-NTA纯化获得的目的蛋白纯度超过90%;SDS-PAGE、Western印迹显示在相对分子质量为36×103位置有目的蛋白的特异条带;非还原电泳及HPLC分析表明,重组蛋白以三聚体的形式存在;ELISA、Octet RED系统同时证实BAFF可与受体TACI结合;增殖实验证实重组蛋白具有生物学活性。结论成功获得纯度超过90%的rhBAFF可溶蛋白,实验证实为具有生物学活性的三聚体结构。
Objective To obtain the extracellular fusion protein of human B cell activating factor (BAFF) and analyze its biological activity. Methods The prokaryotic expression vector pET32a / BAFF was constructed, prokaryotic expressed, affinity purified and identified by SDS-PAGE and Western blotting. The presence of the fusion protein in solution was analyzed by Western blot followed by non-reducing electrophoresis. ELISA, the Octet RED system detects the ability of rhBAFF to bind to the receptor TACI and makes a real-time kinetic analysis. MTS assay rhBAFF promote B cell proliferation activity. Results The fusion protein was highly expressed in soluble form in E. coli BL21. The purity of the target protein was over 90% after purification by Ni-NTA. SDS-PAGE and Western blotting showed that the target protein was specific at the molecular weight of 36 × 103 Band, non-reducing electrophoresis and HPLC analysis showed that the recombinant protein exists in the form of trimer; ELISA, Octet RED system also confirmed that BAFF can bind to the receptor TACI; proliferation experiments confirmed that the recombinant protein has biological activity. Conclusion The rhBAFF soluble protein of more than 90% purity was successfully obtained, which proved to be biologically active trimer structure.