甲状旁腺激素和转铁蛋白N端半分子融合蛋白在毕赤酵母中的表达  

Expression of Fusion Protein of Parathyroid Hormone and Transferrin N-terminal Half-molecule in Pichia pastoris

作　　者：张豪[1] 李晓静[1] 王德解[1] 陈璟[1] 李彦英[1] 李玉玲[1] 沈明山[2] 方宏清[1] 陈惠鹏[1] 

机构地区：[1]军事医学科学院生物工程研究所,北京100071 [2]厦门大学生命科学院,厦门361005

出　　处：《生物工程学报》2005年第5期804-808,共5页Chinese Journal of Biotechnology

基　　金：国家高技术研究与发展项目基金资助(No.2004AA215172)。~~

摘　　要：利用重叠PCR技术将PTH(parathyroidhormone,甲状旁腺激素)基因与TFN(transferrinN_terminalhalf_molecule,转铁蛋白N端半分子)基因在体外融合,融合基因克隆至真核表达载体pPIC9中,转化毕赤酵母GS115。转化子经甲醇诱导后,融合蛋白得到了表达并分泌到发酵上清液中。经SPSepharoseFF阳离子交换层析、PhenylSepharoseFastFlow疏水层析纯化获得了纯度大于95%的PTH_TFN样品。Westernblot分析及腺苷酸环化酶实验证明融合蛋白中的PTH具有与抗PTH抗体结合能力及刺激腺苷酸环化酶的活性,铁饱和实验证明融合蛋白中的TFN和单独的TFN具有相同铁结合能力。因而TFN可望作为PTH的天然运输载体。The fused gene （PTH-TFN） of parathyroid hormone （PTH） gene and transferring N-terminal half-molecule （TFN） gene was amplified by multiple PCR and inserted into pPIC9 vector. The recombinant plasmid pPIC9-PTH-TFN was transformed into Pichia pastoris GS115 by PEG. After methanol induction, the target protein was expressed in fermentation supematant at high level. The fused protein PTH-TFN with purity being higher than 95 % was finally obtained after purification through two-step chromatography ： SP Sepharose Fast Flow and Phenyl Sepharose Fast Flow. Westem blot analysis and adenylate cyclase assay proved that the fused protein exhibited the bioactivity to stimulate cAMP synthesis and the ability to bind Fe^3＋ in the Fe^3＋ saturation study as the recombinant TFN did indicating that TFN could be used as the transcellar carrier of PTH.

关 键 词：甲状旁腺激素 转铁蛋白N端半分子融合蛋白 毕赤酵母 基因表达 运输载体 腺苷酸环化酶 

分 类 号：R346[医药卫生—基础医学]