小C肽人胰岛素原在甲醇酵母（Pichia pastoris）中的分泌表达

发布时间：2018-06-23 00:12

本文选题：小C肽人胰岛素原 + 甲醇酵母Pichia　；参考：《浙江大学》2005年硕士论文

【摘要】：作为治疗糖尿病的药物,重组人胰岛素基因曾先后在大肠杆菌和酿酒酵母S. cerevisiae中表达,但由于大肠杆菌和酿酒酵母自身的缺陷,表达量受到限制。甲醇酵母Pichia pastoris是近年发展起来的真核表达系统,胰岛素原基因已在Pichia pastoris中进行了成功的分泌表达。由于其表达量高,具有翻译后成熟的蛋白修饰等优点而日益受到人们的重视。将小C肽人胰岛素原(B-Arg-Arg-Lys-Arg-A)基因插入到甲醇酵母Pichia pastoris的分泌表达质粒pPIC9中,得到含胰岛素基因的分泌表达质粒I-pPIC9并电转化p.pastoris GSll5(his4)。筛选出整合型His~+Mut~+菌株,然后进一步筛选得到高拷贝转化子。经1L摇瓶培养后,SDS-PAGE证明重组人胰岛素在甲醇酵母中能有效分泌表达,表达量可达到50mg/ml。将发酵液收集后用硫酸铵沉淀、DEAE Sephadex A-25离子柱层析等方法对目的蛋白进行纯化,可使产物中的重组人胰岛素含量由最初的15%提高到30%。胰岛素放射免疫实验,证明重组人胰岛素具有与胰岛素标样相同的抗体结合能力;HPLC 实验证明重组人胰岛素具有与胰岛素标样基本相同的保留时间;小鼠活性实验证明重组人胰岛素具有胰岛素活性。
[Abstract]:As a drug to treat diabetes, recombinant human insulin gene has been expressed in Escherichia coli and Saccharomyces cerevisiae S. cerevisiae. However, due to the absence of Escherichia coli and Saccharomyces cerevisiae, the expression was limited. Methanol yeast Pichia pastoris is an eukaryotic expression system developed in recent years. Proinsulin gene has been successfully expressed in Pichia pastoris. People pay more and more attention to it because of its high expression and the advantages of mature protein modification after translation. The small C-peptide human proinsulin (B-Arg-Lys-Arg-A) gene was inserted into the secretory expression plasmid pPIC9 of methanol yeast Pichia pastoris. The secretory expression plasmid I-pPIC9 containing insulin gene was obtained and transformed into p.pastoris GSll5 (his4). The integrative His- Mut~ strain was screened, and then high copy transformants were obtained. SDS-PAGE showed that recombinant human insulin could be secreted effectively in methanol yeast, and the expression level could reach 50 mg / ml. After the fermentation broth was collected, the target protein was purified by ammonium sulfate precipitation and DEAE Sephadex A-25 ion column chromatography. The recombinant human insulin content in the product was increased from 15% to 30%. Insulin radioimmunoassay, It is proved that recombinant human insulin has the same antibody binding ability as insulin standard sample. HPLC experiment proves that recombinant human insulin has insulin standard base. The same retention period; Mouse activity test showed that recombinant human insulin had insulin activity.
【学位授予单位】：浙江大学
【学位级别】：硕士
【学位授予年份】：2005
【分类号】：R346

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