小鼠B淋巴细胞活化刺激因子可溶性片段在毕赤酵母中的表达、纯化及其生物学活性的初步鉴定  

Expression of mouse soluble B cell activating factor belonging to the TNF family in Pichia pastoris and purification and biological activity of expressed product

作　　者：欧子强[1] 唐勇[1] 向军俭[1] 吕卫东[1] 林婷婷[1] 

机构地区：[1]暨南大学广东省分子免疫与抗体工程重点实验室,广东广州510632

出　　处：《中国生物制品学杂志》2013年第6期787-791,共5页Chinese Journal of Biologicals

基　　金：广东省科技计划项目(2011B020314005)

摘　　要：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 soluble B lymphocyte activating factor（msBAFF）in Pichia pastoris,purify the expressed product and determine its biological activity.Methods Peripheral blood mononuclear cells of BALB/c were separated,from which msBAFF gene was amplified by RT-PCR and inserted into expression vector pPICZαA.The constructed recombinant plasmid pPICZαA-msBAFF was transformed to P.pastoris GS115 and induced with methanol.The expressed recombinant msBAFF protein was purified by ammonium sulfate precipitation and Q Sepharose XL anion exchange column chromatography.The splenic B lymphocytes of BALB/c mice were treated by purified msBAFF alone or combined with anti-IgM,and determined for activity.Results Restriction analysis proved that recombinant plasmid pPICZαA-msBAFF was constructed correctly.The relative molecular mass of expressed recombinant msBAFF was about 20 000,and the expression level reached the peak value 60 h after induction.Purified msBAFF was obtained by ammonium sulfate precipitation,dialysis and Q Sepharose XL anion exchange column chromatography,of which the yield was 20 mg/L.Recombinant BAFF enhanced the activity of mouse B lymphocytes.Conclusion Recombinant msBAFF with high biological activity was successfully expressed in P.pastoris and purified,which laid a foundation of further study on function of mouse BAFF gene and preparation of BAFF-based adjuvant and monoclonal antibody.

关 键 词：B淋巴细胞活化刺激因子 小鼠 毕赤酵母 基因表达 纯化 生物活性 

分 类 号：R392.11[医药卫生—免疫学] Q786[医药卫生—基础医学]