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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。
PTH (parathyroid hormone) gene and TFN (transferrinN_terminalhalf_molecule) were fused in vitro by overlap PCR. The fusion gene was cloned into eukaryotic expression vector pPIC9 and transformed into Pichia pastoris GS115. After the transformants were induced by methanol, the fusion protein was expressed and secreted into the fermentation supernatant. The PTH_TFN samples with more than 95% purity were obtained by SPS Sepharose FF cation exchange chromatography and PhenylSepharoseFast Flow hydrophobic chromatography. Western blot analysis and adenylyl cyclase test showed that the PTH in the fusion protein has the ability to bind to anti-PTH antibody and stimulate adenylate cyclase, and the iron saturation experiment shows that the TFN in the fusion protein has the same iron Combine ability. TFN is therefore expected to serve as a natural transport vector for PTH.