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  本文選題：人血清白蛋白　切入點：M2e　出處：《吉林大學》2010年博士論文　論文類型：學位論文

【摘要】： M2 (Matrix protein 2,M2)蛋白是流感病毒主要的膜蛋白之一。研究發(fā)現(xiàn),針對M2蛋白的單克隆抗體在細胞培養(yǎng)中具有抑制流感病毒復制的功能。進一步證明,M2蛋白胞外功能區(qū)(Extracellular domain of M2 protein,M2e)誘導機體產(chǎn)生的抗體同樣具有保護性。目前,在人群中流行的所有甲型流感病毒,其M2蛋白的胞外區(qū)都未發(fā)現(xiàn)存在明顯差異,認為M2e在流感病毒間是高度保守的。因此,M2e蛋白被認為是可刺激機體對不同流感變異株都產(chǎn)生具有交叉保護效果的保護性抗原。鑒于此,本研究圍繞M2e蛋白進行了如下工作。 利用基因重組技術(shù)構(gòu)建了畢赤酵母真核表達載體pPICZαC-HSA/M2e,電轉(zhuǎn)化畢赤酵母X-33,SDS-PAGE篩選出能夠穩(wěn)定高效分泌表達重組HSA/M2e的工程菌。重組蛋白經(jīng)AKTA explorer多功能純化系統(tǒng)純化,Western Blot方法進一步證明表達的重組HSA/M2e分別具有HSA和M2e的活性;蛋白質(zhì)N-端測序證明表達的重組HSA/M2e與天然HSA N-端氨基酸序列相同。利用80 L發(fā)酵罐進行HSA/M2e中試規(guī)模發(fā)酵,并對其發(fā)酵條件進行優(yōu)化,HSA/M2e表達量可達到1080 mg/L,同時建立了一種分離純化HSA/M2e的新方法。 用純化的融合蛋白HSA/M2e免疫BALB/c小鼠三次后,用甲型流感病毒H1N1和H3N2對小鼠滴鼻進行攻擊,從小鼠的肺部病毒滴度、體重變化及死亡率等幾個方面檢測M2e蛋白的免疫效果。結(jié)果表明,融合蛋白HSA/M2e能顯著減少小鼠體重的丟失和降低小鼠的死亡率,保護小鼠抵抗甲型流感病毒H1N1和H3N2的攻擊。 本研究創(chuàng)新之處:1)首次在畢赤酵母中高效表達了融合蛋白HSA/M2e;2)利用80 L發(fā)酵罐進行了HSA/M2e畢赤酵母工程菌的中試規(guī)模發(fā)酵,對其表達條件進行了優(yōu)化,建立了一種適用于大規(guī)模分離純化HSA/M2e的方法,證明應用畢赤酵母表達系統(tǒng)表達的重組蛋白HSA/M2e具有HSA和M2e的活性;3)融合蛋白HSA/M2e能顯著地減少小鼠體重的丟失和降低小鼠的死亡率,并降低肺部病毒滴度,保護小鼠抵抗甲型流感病毒的攻擊。
[Abstract]:The M2 Matrix protein 2 (M2) protein is one of the major membrane proteins of influenza viruses. The monoclonal antibody against M2 protein can inhibit the replication of influenza virus in cell culture. It is further proved that the antibody induced by extracellular domain of M2 protein M2e is also protective. No significant difference in the extracellular domain of M2 protein was found in all influenza A viruses in the population. It is considered that M2e is highly conserved among influenza viruses. Therefore, M2e protein is considered to be a protective antigen that stimulates the body to produce cross-protective effects on different influenza variants. In view of this, this study has carried out the following work around M2e protein. The eukaryotic expression vector pPICZ 偽 C-HSA-M2eof Pichia pastoris was constructed by gene recombination technique, and the recombinant protein was purified by AKTA explorer multifunctional purification system. The recombinant protein was isolated from Pichia pastoris X-33 and SDS-PAGE. The recombinant protein was purified by AKTA explorer multifunctional purification system. Methods it was further proved that the expressed recombinant HSA/M2e had the activity of HSA and M2e, respectively. The sequence of the expressed recombinant HSA/M2e was the same as that of the natural HSA. The recombinant HSA/M2e was fermentated on a pilot scale with 80 L fermenter, and the amino acid sequence of the recombinant HSA/M2e was the same as that of the natural HSA. The expression of HSA / M2e reached 1080 mg / L, and a new method was established to isolate and purify HSA/M2e. After immunizing BALB/c mice with purified fusion protein HSA/M2e for three times, influenza A (H1N1) and H3N2 were used to intranasally attack the mice. The results showed that the fusion protein HSA/M2e could significantly reduce the weight loss and mortality of mice and protect mice against influenza A (H1N1) and H3N2 attacks. The innovation of this study was: (1) the fusion protein HSA / M2e2 was expressed efficiently in Pichia pastoris for the first time. The medium scale fermentation of Pichia pastoris HSA/M2e was carried out in 80L fermenter, and the expression conditions were optimized. A method for large-scale isolation and purification of HSA/M2e was established. It was proved that the recombinant protein HSA/M2e expressed by Pichia pastoris had the activity of HSA and M2e. The fusion protein HSA/M2e could significantly reduce the loss of body weight and the mortality of mice. And reduce the lung virus titer, protect mice against influenza A virus attack.
【學位授予單位】：吉林大學
【學位級別】：博士
【學位授予年份】：2010
【分類號】：R373

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