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目的在毕赤酵母中表达小鼠B淋巴细胞活化刺激因子(B cell activating factor belonging to the TNF family,BAFF)可溶性片段(msBAFF),纯化后检测其生物学活性。方法采用RT-PCR法从BALB/c小鼠外周血单核细胞中扩增msBAFF基因,插入表达载体pPICZαA中,构建重组表达质粒pPICZαA-msBAFF,转化巴斯德毕赤酵母GS115,甲醇诱导表达。表达的重组msBAFF蛋白经硫酸铵沉淀及Q Sepharose XL阴离子交换柱纯化后,分别单独及与anti-IgM共同作用于BALB/c小鼠脾脏B淋巴细胞,检测其对B淋巴细胞活力的影响。结果重组表达质粒pPICZαA-msBAFF经双酶切鉴定构建正确;表达的重组msBAFF蛋白相对分子质量约为20 000,诱导60 h表达量较高;经硫酸铵沉淀、透析除盐及Q Sepharose XL阴离子交换柱纯化,获得了较纯的msBAFF,BCA法测定纯化蛋白的产率为20 mg/L;重组BAFF蛋白具有促进小鼠B淋巴细胞活力的作用。结论成功在毕赤酵母中表达了重组msBAFF蛋白,纯化的重组蛋白具有良好的生物学活性,为进一步研究小鼠BAFF基因的功能及基于BAFF的佐剂与单克隆抗体的制备奠定了物质基础。
OBJECTIVE: To express mouse plasma membrane factor B (BAFF) soluble fragment (msBAFF) in Pichia pastoris and determine its biological activity after purification. Methods msBAFF gene was amplified from peripheral blood mononuclear cells of BALB / c mice by RT-PCR and inserted into expression vector pPICZαA. The recombinant plasmid pPICZαA-msBAFF was transformed into Pichia pastoris GS115 and induced by methanol. The expressed recombinant msBAFF protein was purified by ammonium sulfate precipitation and Q Sepharose XL anion exchange column, and the effect of anti-IgM on the B lymphocyte activity of BALB / c mice was tested separately. Results The recombinant plasmid pPICZαA-msBAFF was identified by double enzyme digestion. The expressed recombinant protein msBAFF had a relative molecular mass of about 20 000 and a high expression level at 60 h after induction. Ammonium sulfate precipitation, desalination and Q Sepharose XL anion exchange Column purification, the pure msBAFF was obtained. The yield of purified protein was 20 mg / L as determined by BCA method. Recombinant BAFF protein could promote the activity of B lymphocytes in mice. Conclusions Recombinant msBAFF protein was successfully expressed in Pichia pastoris. The purified recombinant protein has good biological activity, which lays the material foundation for further study on the function of mouse BAFF gene and the preparation of BAFF-based adjuvant and monoclonal antibody.