甲型流感病毒M2蛋白的表达、纯化及其免疫原性的研究

发布时间：2018-09-10 06:35

【摘要】：流感病毒可在短期内致病并迅速蔓延,其中甲型流感病毒的流行性最强,几乎每年都会在世界范围内引发不同程度的流行,危及人类健康并且造成严重的经济损失。如今大多数甲型流感疫苗为病毒裂解疫苗、灭活疫苗或针对病毒表面抗原HA和NA的重组疫苗,但是由于表面抗原HA和NA变异很大,这些疫苗在不同亚型间的交叉保护作用受到很大的局限。M2蛋白是流感病毒颗粒表面的第三种抗原蛋白,在所有已知的不同亚型的甲型流感病毒中高度保守,自1918年流感大流行以来几乎未发生变化。因此,M2蛋白成为了一个甲型流感广谱疫苗的理想候选抗原。 M2蛋白在病毒颗粒包膜上呈低密度表达,灭活病毒疫苗很难诱导出M2特异性抗体,因此低成本的原核表达系统成为获得大量抗原的有效途径。但是,M2重组蛋白的疏水性强,在原核系统中难以获得大量的表达。我们在尝试原核表达M2蛋白的过程中,意外地获得M2蛋白突变体,在C端一段的短肽,使其在E.coli中具有很高的表达量。我们将其重新改造构建后命名为M2MH,经过摸索和优化纯化条件,得到了大量较高纯度的重组蛋白M2MH,质谱和电镜结果表明M2MH形成了多聚体,提示其作为抗原,具备很好的免疫原性。用纯化的重组蛋白M2MH免疫BALB/C小鼠后,在小鼠体内诱导出高滴度的M2特异性多克隆抗血清,不仅能够识别真核细胞表面表达的重组蛋白M2,还能够识别甲型流感不同亚型的病毒表面抗原蛋白M2,以及被流感病毒侵染的MDCK细胞表面表达的天然蛋白M2,说明M2MH虽有突变多肽,却保有了很强的免疫原性。致死量的甲型流感病毒对免疫后的小鼠滴鼻攻击,从小鼠的体重变化、生存率变化、肺组织病变程度几个方面评估重组蛋白的免疫保护作用。结果表明重组蛋白能够在动物体内诱导出一定的免疫反应,使小鼠有效抵抗病毒的攻击,该实验结果为进一步利用M2MH作为抗原研制广谱疫苗提供了依据。另外,我们运用杂交瘤技术,成功筛选出28株稳定分泌M2蛋白特异性的单克隆抗体的杂交瘤细胞株,为甲型流感病毒感染的诊断、治疗及病毒致病机制研究奠定了基础。
[Abstract]:Influenza virus can cause disease and spread rapidly in a short period of time. The influenza A virus is the most prevalent in the world almost every year, it will cause different degrees of epidemic in the world, endanger human health and cause serious economic losses. Today, most influenza A vaccines are viral cleavage vaccines, inactivated vaccines or recombinant vaccines targeting virus surface antigens HA and NA, but because of the large variation in surface antigens HA and NA, The cross-protection of these vaccines between different subtypes is greatly limited. M2 protein is the third antigen protein on the surface of influenza virus particles and is highly conserved in all known subtypes of influenza A viruses. Little has changed since the 1918 influenza pandemic. Therefore, M 2 protein has become an ideal candidate antigen for influenza A wide spectrum vaccine. M 2 protein is expressed in low density on the coating of virus particles, so it is difficult to induce M2 specific antibody by inactivated virus vaccine. Therefore, low-cost prokaryotic expression system has become an effective way to obtain a large number of antigens. However, the M 2 recombinant protein is so hydrophobic that it is difficult to express in prokaryotic system. In the process of prokaryotic expression of M2 protein, we accidentally obtained the mutant M2 protein, a short peptide in C-terminal, which makes it have a high expression level in E.coli. The recombinant protein M2MH with high purity was obtained by optimizing the purification conditions. The results of mass spectrometry and electron microscopy showed that M2MH formed a polymer, which indicated that it was an antigen and had good immunogenicity. After immunizing BALB/C mice with purified recombinant protein M2MH, a high titer M2 specific polyclonal antiserum was induced in mice. It can recognize not only the recombinant protein M2 expressed on the surface of eukaryotic cells, but also the virus surface antigen protein M2 of different subtypes of influenza A, and the natural protein M2 expressed on the surface of MDCK cells infected by influenza virus, indicating that M2MH has mutated polypeptides. But it retains a strong immunogenicity. The immune protective effect of recombinant protein was evaluated from the changes of body weight, survival rate and lung tissue pathological changes in mice after immunization with lethal influenza A virus. The results showed that the recombinant protein could induce a certain immune response in animals and make mice resist the attack of virus effectively. The results provided a basis for the further development of wide-spectrum vaccine using M2MH as antigen. In addition, we successfully screened 28 hybridoma cell lines secreting monoclonal antibody against M2 protein by using hybridoma technique, which laid a foundation for the diagnosis, treatment and pathogenesis of influenza A virus infection.
【学位授予单位】：清华大学
【学位级别】：硕士
【学位授予年份】：2010
【分类号】：R392

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