重组融合人血清白蛋白-人白介素-2 C125A突变体在毕赤酵母中的表达

Expression of the Fusion Protein Human Serum Album/Mutant Human Interleukin 2C125A in Pichia pastoris

DOI：

中文关键词: 突变型人白介素-2 人血清白蛋白 毕赤酵母 融合蛋白

英文关键词: mutant Human interleukin-2 Human serum album Pichia pastoris fusion protein

基金项目:国家自然科学基金项目(30970029);江苏省产学研联合创新资金计划项目(BY2009110)

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中文摘要:

构建编码人血清白蛋白-人白介素-2 C125A的重组表达质粒,在Pichia pastorisGS115中表达,获得具有较高IL-2活性的融合蛋白。设计并合成符合P.pastoris密码子偏好,且含有C125A突变的IL-2编码基因IL-2m,通过酶切连接方法将其与人血清白蛋白(HSA)的编码基因连接为融合蛋白HSA-IL2m的编码基因。克隆到表达质粒pPIC9K中,电击转化P.pastorisGS115感受态细胞,获得重组P.pastorisGS115/pPHIm基因工程菌。摇瓶发酵获得分泌表达产物。结果:Western blot鉴定结果显示该融合蛋白与IL-2、HSA的抗体都能发生免疫反应。经脱盐、冻干制备的粗蛋白的IL-2生物学活性为1.51×106IU/mg。结论:在P.pastorisGS115中成功表达了具有人白介素-2生物学活性的HSA-IL2m融合蛋白。

英文摘要:

The aim of this study was to obtain the fusion protein HSA-IL2m with high IL2 bioactivity,for this,the recombinant plasmid encoding HSA-IL2C125A was constructed and expressed in Pichia pastoris GS115.The DNA fragment encoding mutant IL-2 of C125A was design and synthesized based on codon usage bias of P.pastoris.The fragment was spliced with Human Serum Album encoding gene and then the fusion gene was cloned into pPIC9K to construct the expression plasmid pPHIm by restrictive digestion and ligation.Recombinant yeast of P.pastoris GS115/pPHIm was obtained with pPHIm linearization and electroporation.The excreting product was obtained under shake flask culture.Results: Western-blot showed the fusion protein reactive positively with the antibody of IL-2 and HSA,respectively.The crude fusion protein prepared by desalting and freeze-drying showed IL-2 bioactivity of 5.0 105 IU/mg.The results demonstrated that the fusion protein of HSA-IL2m with IL-2 bioactivity was successfully expressed in P.pastoris GS115.

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