摘要
目的 为深入研究趋化因子新变异体MPIF 1β的结构与功能 ,利用pKPL3a MPIF 1β表达质粒进行原核表达、纯化工艺研究及活性检测。方法 MPIF 1β重组蛋白以包涵体形式存在 ,占总菌体蛋白的 15 % ,工程菌经超声破碎 ,包涵体洗涤 ,变性、复性、肝素SepharoseCL 6B亲和层析 ,CMSepharoseFastFlow和SephacrylS 2 0 0层析分离获得目的蛋白。结果 还原SDS PAGE分析相对分子质量 15 0 0 0u ,纯度 99.0 % ,纯化倍数 6 .6倍 ,总回收率 9.0 7%。体外生物学活性检测证明MPIF 1β重组蛋白对小鼠骨髓集落形成具有抑制作用 ,对U937细胞具有趋化活性 ,且呈一定的剂量依赖关系。结论 建立了简便可行的MPIF 1β重组蛋白纯化路线 ,初步证明了MPIF
Objective In order to study the structure and function of MPIF 1β, a new isoform of human MPIF 1β, recombinant protein was expressed in E.coli and its purifying procedure was determined. Methods It was found that the recombinant protein presented as inclusion body and had an expressing level up to 15%. For the purification of the MPIF 1β, the inclusion body was washed, denatured and renatured, and then three steps of chromatography (heparin affinity chromatography, CM ion exchange and Sephacryl S 200 size exclusion) were carried out to obtain purified protein. Results SDS PAGE analysis showed a single band at molecular weight 15 000 u. The purity of target protein was up to 99.0%, the total recovery rate is 9.07%. Bioassay in vitro showed MPIF 1β could inhibit mouse LPP CFC and attract U937 cells with dose dependent manner. Conclusions A simply method of purified recombinant MPIF 1β protein is established and its biological activity has been found.
出处
《免疫学杂志》
CAS
CSCD
北大核心
2002年第4期311-314,共4页
Immunological Journal
