本文選題：小C肽人胰島素原 + 甲醇酵母Pichia　； 參考：《浙江大學(xué)》2005年碩士論文

【摘要】：作為治療糖尿病的藥物,重組人胰島素基因曾先后在大腸桿菌和 釀酒酵母S. cerevisiae中表達(dá),但由于大腸桿菌和釀酒酵母自身的缺 陷,表達(dá)量受到限制。甲醇酵母Pichia pastoris是近年發(fā)展起來的真 核表達(dá)系統(tǒng),胰島素原基因已在Pichia pastoris中進(jìn)行了成功的分泌 表達(dá)。由于其表達(dá)量高,具有翻譯后成熟的蛋白修飾等優(yōu)點而日益受 到人們的重視。 將小C肽人胰島素原(B-Arg-Arg-Lys-Arg-A)基因插入到甲醇 酵母Pichia pastoris的分泌表達(dá)質(zhì)粒pPIC9中,得到含胰島素基因的 分泌表達(dá)質(zhì)粒I-pPIC9并電轉(zhuǎn)化p.pastoris GSll5(his4)。篩選出整 合型His~+Mut~+菌株,然后進(jìn)一步篩選得到高拷貝轉(zhuǎn)化子。經(jīng)1L搖瓶 培養(yǎng)后,SDS-PAGE證明重組人胰島素在甲醇酵母中能有效分泌表 達(dá),表達(dá)量可達(dá)到50mg/ml。將發(fā)酵液收集后用硫酸銨沉淀、DEAE Sephadex A-25離子柱層析等方法對目的蛋白進(jìn)行純化,可使產(chǎn)物中 的重組人胰島素含量由最初的15%提高到30%。 胰島素放射免疫實驗,證明重組人胰島素具有與胰島素標(biāo)樣相同 的抗體結(jié)合能力;HPLC 實驗證明重組人胰島素具有與胰島素標(biāo)樣基 本相同的保留時間;小鼠活性實驗證明重組人胰島素具有胰島素活 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.
【學(xué)位授予單位】：浙江大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2005
【分類號】：R346

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相關(guān)期刊論文 前2條

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