流感病毒M2e蛋白的制备及其抗甲型流感病毒研究

发布时间：2018-03-20 21:52

本文选题：人血清白蛋白　切入点：M2e　出处：《吉林大学》2010年博士论文　论文类型：学位论文

【摘要】： M2 (Matrix protein 2,M2)蛋白是流感病毒主要的膜蛋白之一。研究发现,针对M2蛋白的单克隆抗体在细胞培养中具有抑制流感病毒复制的功能。进一步证明,M2蛋白胞外功能区(Extracellular domain of M2 protein,M2e)诱导机体产生的抗体同样具有保护性。目前,在人群中流行的所有甲型流感病毒,其M2蛋白的胞外区都未发现存在明显差异,认为M2e在流感病毒间是高度保守的。因此,M2e蛋白被认为是可刺激机体对不同流感变异株都产生具有交叉保护效果的保护性抗原。鉴于此,本研究围绕M2e蛋白进行了如下工作。利用基因重组技术构建了毕赤酵母真核表达载体pPICZαC-HSA/M2e,电转化毕赤酵母X-33,SDS-PAGE筛选出能够稳定高效分泌表达重组HSA/M2e的工程菌。重组蛋白经AKTA explorer多功能纯化系统纯化,Western Blot方法进一步证明表达的重组HSA/M2e分别具有HSA和M2e的活性;蛋白质N-端测序证明表达的重组HSA/M2e与天然HSA N-端氨基酸序列相同。利用80 L发酵罐进行HSA/M2e中试规模发酵,并对其发酵条件进行优化,HSA/M2e表达量可达到1080 mg/L,同时建立了一种分离纯化HSA/M2e的新方法。用纯化的融合蛋白HSA/M2e免疫BALB/c小鼠三次后,用甲型流感病毒H1N1和H3N2对小鼠滴鼻进行攻击,从小鼠的肺部病毒滴度、体重变化及死亡率等几个方面检测M2e蛋白的免疫效果。结果表明,融合蛋白HSA/M2e能显著减少小鼠体重的丢失和降低小鼠的死亡率,保护小鼠抵抗甲型流感病毒H1N1和H3N2的攻击。本研究创新之处:1)首次在毕赤酵母中高效表达了融合蛋白HSA/M2e;2)利用80 L发酵罐进行了HSA/M2e毕赤酵母工程菌的中试规模发酵,对其表达条件进行了优化,建立了一种适用于大规模分离纯化HSA/M2e的方法,证明应用毕赤酵母表达系统表达的重组蛋白HSA/M2e具有HSA和M2e的活性;3)融合蛋白HSA/M2e能显著地减少小鼠体重的丢失和降低小鼠的死亡率,并降低肺部病毒滴度,保护小鼠抵抗甲型流感病毒的攻击。
[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 伪 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.
【学位授予单位】：吉林大学
【学位级别】：博士
【学位授予年份】：2010
【分类号】：R373

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