本文選題：基質(zhì)金屬蛋白酶 + 整合素　； 參考：《生物技術(shù)》2017年03期

【摘要】：[目的]構(gòu)建rSalmosin-Mel融合蛋白畢赤酵母真核表達(dá)載體,建立表達(dá)純化工藝并初步評價其抗腫瘤活性。[方法]rSalmosin-Mel基因同pPIC9K相連,構(gòu)建pPIC9K/rD-M真核表達(dá)載體,電轉(zhuǎn)化至畢赤酵母菌GS115中。對重組菌的發(fā)酵時間、甲醇濃度及培養(yǎng)基pH進(jìn)行優(yōu)化。采用QSepharose HP和Sephadex G-75層析技術(shù)純化重組蛋白rD-M。通過MTT比色法檢測rD-M對MCF-7細(xì)胞增殖的抑制作用。[結(jié)果]獲得了高效分泌表達(dá)rD-M融合蛋白的酵母工程菌株,確定了在28℃、pH 6.0、濃度為1.5%甲醇誘導(dǎo)96 h發(fā)酵條件。通過層析純化出純度大于95%的重組蛋白。MTT實驗結(jié)果表明,0.25、0.5、1、2和4 nmol/L的rD-M對MCF-7細(xì)胞增殖具有明顯的抑制作用。[結(jié)論]實現(xiàn)了rSalmosin-Mel融合蛋白在畢赤酵母中最優(yōu)條件下的分泌型表達(dá),為其作為基因工程抗腫瘤藥物奠定基礎(chǔ)。
[Abstract]:[objective] to construct the eukaryotic expression vector of rSalmosin-Mel fusion protein Pichia pastoris, to establish the expression and purification process and to evaluate the antitumor activity of rSalmosin-Mel fusion protein. [methods] the rSalmosin-Mel gene was linked to pPIC9K, and the eukaryotic expression vector pPIC9K / rD-M was constructed and electrotransformed into Pichia pastoris GS115. The fermentation time, methanol concentration and pH of culture medium were optimized. The recombinant protein was purified by QSepharose HP and Sephadex G-75 chromatography. The inhibitory effect of rD-M on the proliferation of MCF-7 cells was detected by MTT colorimetry. [results] yeast engineering strain secreting and expressing rD-M fusion protein was obtained, and the fermentation conditions were determined at 28 鈩，

本文編號：1994237