人甲状旁腺激素-血清白蛋白融合蛋白的构建及在毕赤酵母中的表达

利用重叠PCR技术拼接PTH和HSA基因，并将构建好的融合基因插入到载体pUC19测序后插入表达载体pPIC9K中，在启动子AOXⅠ和α交配因子信号肽的作用下，分泌表达融合蛋白PTH-HSA。重组质粒pPIC9K/PTH-HSA经SalⅠ线性化后,电击转化毕赤酵母KM71,经G418筛选得到的转化子。PCR鉴定后,用甲醇诱导表达,蛋白电泳分析表明融合基因得到表达; Western blot分析表明发酵液上清中表达的融合蛋白PTH-HSA具有HSA的抗原性:用酶标法测定发酵上清中融合蛋白的甲状旁腺激素活性为318IU/ml

Secretory expression of the fusion protein PTH-HSA in Pichia pastoris

The fused gene (PTH-HSA)of parathyroid hormone (PTH) gene and Human Serum Albumin(HSA)gene was amplified by Overlapping PCR technology. The spliced gene was clone into Pichia pastoris secretory vector pPIC9K. With the help of promoter AOX1 and mat α signal peptide, the PTH-HSA gene was designed to secretory expression。Linearized by restriction enzyme SalI, The recombinant plasmid pPIC9K/PTH-HSA was transformed into Pichia pastoris KM71 by electroporation. The recombinant strains which were identified by G418 and PCR analysis, were induced by methanol to express protein PTH-HSA. the target protein was expressed in fermentation supernatant. Western blot analysis of the fusion protein showed that the expressed fusion protein PTH-HSA had the antigenicity of HSA.adenylate cyclase assay proved that the fused protein exhibited the bioactivity to stimulate cAMP synthesis The specific activity of broth was about 318IU/ml 。