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目的:构建人B淋巴细胞刺激因子(hBAFF)的真核表达质粒,表达具有生物学功能的Fc-hBAFF融合蛋白。方法:PCR扩增hbaff胞外区编码基因,加入HA信号肽与hIgG1 Fc后,连入pTT3真核表达载体,采用磷酸钙法转染293T细胞。培养上清经蛋白G纯化后,SDS-PAGE与Westernblot鉴定目的蛋白的表达。流式细胞术检测Fc-hBAFF对人B细胞的增殖情况。结果:成功构建pTT3-HA-hIgG1 Fc-hBAFF融合表达质粒,体外大量表达纯化的Fc-hBAFF蛋白经SDS-PAGE与Western blot鉴定正确,并发现Fc-hBAFF蛋白对人外周血B细胞有明显促增殖作用。同时通过尾静脉高压注射的方式证实该质粒能在体内持续表达7 d。结论:体外表达并获得了具有生物学功能的Fc-hBAFF融合蛋白。
Objective: To construct eukaryotic expression plasmid of human B lymphocyte stimulating factor (hBAFF) and express Fc-hBAFF fusion protein with biological function. Methods: The coding region of extracellular domain of hbaff was amplified by PCR. After HA signal peptide and hIgG1 Fc were added, it was inserted into pTT3 eukaryotic expression vector and transfected into 293T cells by calcium phosphate method. After the culture supernatant was purified by protein G, the expression of the target protein was identified by SDS-PAGE and Western blot. Flow cytometry was used to detect the proliferation of human B cells by Fc-hBAFF. Results: The recombinant plasmid pTT3-HA-hIgG1 Fc-hBAFF was successfully constructed. The purified Fc-hBAFF protein expressed in vitro was identified by SDS-PAGE and Western blot. Fc-hBAFF protein was significantly enhanced on human peripheral blood B cells Proliferation effect. At the same time, high-pressure injection through the tail vein confirmed that the plasmid sustained expression in vivo 7 d. Conclusion: The Fc-hBAFF fusion protein with biological function was expressed in vitro.