重组人B细胞激活因子融合蛋白的表达、纯化及活性鉴定  

Expression,purification and bioactivity identification of recombinant human BAFF fusion protein

作　　者：王茹[1,2] 林周[1] 魏化伟[1] 彭辉[1] 侯春梅[1] 冯健男[1] 沈倍奋[1] 黎燕[1] 

机构地区：[1]军事医学科学院基础医学研究所免疫学研究室,北京100850 [2]解放军第305医院检验科,北京100017

出　　处：《军事医学》2013年第2期91-96,共6页Military Medical Sciences

基　　金：国家重大新药创制科技重大专项资助项目(20102X09401-407);国家863计划重大项目(2012AA02A302)

摘　　要：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 human B cell activating factor（BAFF） extracellular domain fusion protein and analyze its biological activity. Methods pET32a/BAFF prokaryotie expression vector for prokaryotic expression was constructed. Affinity purification and identification were conducted by SDS-PAGE and by Western blotting. The form of fusion protein in solution was analyzed by Western blotting after non-reducing electrophoresis and HPLC. ELISA was used to detect the bind- ing ability of rhBAFF interaction with TACI and the real-time kinetic analysis was made by Octet RED system. The activity of rhBAFF promoting B-cell proliferation was analyzed by MTS assay. Results The fusion protein was expressed in E. coli BL21 as soluble protein with high efficiency and purified by Ni-NTA with a purity of more than 90%. The recombinant pro- tein with a relative molecular mass of about 36 kDa could specifically recognize the antibody to BAFF. Non-reducing electro- phoresis and HPLC analysis showed that the recombinant protein adopted typical trimeric structures. ELISA, Octet RED system analysis confirmed that BAFF could bind TACI with high affinity. MTS assay confirmed that the recombinant protein could activate the B cell proliferation. Conclusion High-purity rhBAFF soluble protein of trimer structure with high biolog- ical activity is successfully obtained.

关 键 词：B细胞激活因子 受体 三聚体 相互作用 细胞增殖 表达纯化 

分 类 号：R392.1[医药卫生—免疫学]