本文關(guān)鍵詞：巨噬細(xì)胞激活劑Polytuftsin在畢赤酵母中的表達(dá)及其活性檢測　出處：《中國協(xié)和醫(yī)科大學(xué)》2009年碩士論文　論文類型：學(xué)位論文

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【摘要】： 目的: 構(gòu)建可持續(xù)分泌表達(dá)巨噬細(xì)胞激活劑Polytuftsin蛋白的重組畢赤酵母菌株,鑒定所獲得的Polyttuftsin純化重組蛋白的生物學(xué)活性。 方法: 合成串聯(lián)編碼40個Tuftsin單體以及6His純化標(biāo)簽的載體質(zhì)粒pPIC9K-6His-Polytuftsin;PEG1000化學(xué)法將線性化后的載體質(zhì)粒轉(zhuǎn)化入畢赤酵母菌GS115中,篩選鑒定獲得高拷貝陽性轉(zhuǎn)化子;甲醇培養(yǎng)基誘導(dǎo)其分泌表達(dá)Polytuftsin蛋白,親和純化蛋白粗品后鑒定最終得到的純化蛋白:體內(nèi)、外水平上分別檢測純化后的Polytuftsin重組蛋白的生物學(xué)功能。 結(jié)果: 1)成功構(gòu)建了可持續(xù)分泌表達(dá)Polytuftsin蛋白的重組畢赤酵母菌株,并證實經(jīng)親和純化后得到的Polytuftsin重組蛋白是正確的目的產(chǎn)物,分子量約為20Kda; 2)純化后的Polytuftsin重組蛋白能夠提高鼠源巨噬細(xì)胞分泌NO、TNF-α因子的能力,較對照組相比,提高比率可達(dá)到43%;與此同時,還可提高人源巨噬細(xì)胞分泌NO因子能力,較對照組相比,提高比率可達(dá)到56%; 3)純化后的Polytuftsin重組蛋白能夠提高小鼠腹腔巨噬細(xì)胞的殺瘤能力,殺傷率可達(dá)到50%;利用Polytuftsin純化蛋白刺激后的人源巨噬細(xì)胞對人胰腺癌細(xì)胞系JF305的殺傷活力最大,可達(dá)31%; 4)體內(nèi)小鼠L1210白血病模型治療實驗結(jié)果發(fā)現(xiàn):較對照組相比,給予Polytuftsin重組蛋白后的小鼠出腹水時間延遲,且小鼠生存期延長,平均達(dá)到21天。 結(jié)論: 經(jīng)畢赤酵母表達(dá)系統(tǒng)獲得的Polytuftsin重組蛋白,可以有效激活巨噬細(xì)胞,是一種新型的巨噬細(xì)胞蛋白激活劑。
[Abstract]:Objective: A recombinant Pichia pastoris strain expressing macrophage activator Polytuftsin protein was constructed. The biological activity of recombinant protein purified by Polyttuftsin was identified. Methods: The vector plasmid pPIC9K-6His-Polytuftsin encoding 40 Tuftsin monomers and 6His purified label was synthesized. The linearized vector plasmid was transformed into Pichia pastoris GS115 by PEG1000 chemical method, and high copy positive transformants were obtained by screening and identification. Methanol medium induced the secretion and expression of Polytuftsin protein. The purified protein was identified as the final purified protein: in vivo. The biological function of purified Polytuftsin recombinant protein was detected in vitro. Results: 1) A recombinant Pichia pastoris strain expressing Polytuftsin protein was successfully constructed. The recombinant Polytuftsin protein obtained by affinity purification was proved to be the correct target product with a molecular weight of about 20kda. 2) the purified Polytuftsin recombinant protein could enhance the ability of murine macrophages to secrete NO-TNF- 偽, and the ratio of increase was 43% compared with the control group. At the same time, the ability of human macrophages to secrete no factor was increased, and the ratio of increase reached 56% compared with the control group. 3) the purified Polytuftsin recombinant protein could improve the tumor killing ability of murine peritoneal macrophages, and the killing rate could reach 50%. Human macrophages stimulated by purified Polytuftsin protein had the highest killing activity against human pancreatic cancer cell line JF305, up to 31%. 4) the experimental results of L1210 leukemia model in vivo showed that: compared with the control group, the ascites time of mice treated with Polytuftsin recombinant protein was delayed. The survival time of mice was prolonged, reaching 21 days on average. Conclusion: The recombinant Polytuftsin protein obtained by Pichia pastoris expression system can effectively activate macrophages and is a new type of macrophage protein activator.
【學(xué)位授予單位】：中國協(xié)和醫(yī)科大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2009
【分類號】：R329

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