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

用重叠PCR技术将PTH（parathyroid hormone, 甲状旁腺激素）基因与TFN（transferrin N_terminal half_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 SP Sepharose F F阳离子交换层析、Phenyl Sepharose Fast Flow疏水层析纯化获得了纯度大于95％的PTH_TFN样品。Western blot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。

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

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 supernatant 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.Western 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.