【作者】 沈婧；

【导师】 邬开朗；

【作者基本信息】 武汉大学 ， 微生物学， 2022， 硕士

【摘要】 呼吸道病毒是感染人体呼吸系统的主要病毒,这种疾病在人群中广泛流行,引起咳嗽、头痛、疲劳、严重的会导致肺部疾病等相关症状,甚至死亡,因此引起全球人们广泛关注。其中人呼吸道合胞病毒（HRSV）在老年人和婴幼儿等免疫力较低的人群中广泛传播,引起呼吸道感染,造成肺炎和支气管炎等疾病,带来了巨大的损失,RSV的两种结构蛋白F蛋白和G蛋白一起介导病毒与宿主细胞的结合,使得病毒可以进入细胞。RSV F蛋白还具有较强的免疫原性,因此有关的疫苗和药物研发大多关注于RSV F蛋白并将其认为是重要靶点;流感病毒在全球引起过多次大流行,死亡人数高达数千万,流感病毒中的HA蛋白介导病毒与细胞膜的融合,从而使病毒进入细胞进行复制。RSV病毒基因组编码11种蛋白质,其中编码的F蛋白是现在的热门靶点蛋白,它与多种人体内的多种蛋白质相互作用,F蛋白的最初形态被称为F₀,F₀蛋白被切割后产生F₂亚基和F₁亚基进行融合成为一个单位,这样的单位三个聚合在一起形成三聚体,此时F蛋白行使正常功能。本实验合成F蛋白基因后进行进行点突变,在HEK293E和Expi293F中进行分泌表达,通过超滤浓缩后通过与Ni-NTA进行亲和层析,并经过分子筛Superose ^TM6 Increase 10/300 GL凝胶层析后获得稳定且均一的蛋白质进行电镜负染检测,后续我们可以制备与其相互作用的蛋白进行类似的制备方法后进行结合,在冷冻电子显微镜下观察结果,再进行进一步的分析。本研究结果为后续针对病毒F蛋白的抗原性以及免疫原性的研究打下了坚实的基础。流感疫苗大多以鸡胚为基质作为制备工艺进行生产,然而鸡胚具有数量限制并且在生产过程中很有可能发生污染,因此以细胞为基质的流感疫苗也逐渐开始研发,其中MDCK细胞被认为是流感病毒的敏感细胞,目前已有通过MDCK细胞为基质制备的流感疫苗。在不同的基质中,流感病毒的HA蛋白可能在结构和数量上会有所不同,而HA蛋白在流感病毒的感染过程中起到重要作用,不同结构的HA蛋白是否会影响疫苗的作用,我们仍然需要进一步探究。本实验中我们分别在鸡胚和MDCK细胞中扩增H3N2病毒,并将过滤后的病毒液进行蔗糖密度梯度离心纯化,进行病毒核酸PCR检测后再在经过电镜负染观察,发现通过鸡胚扩增的病毒颗粒与通过MDCK细胞扩增的病毒颗粒大小和形态略有不同,鸡胚扩增出的纯化后病毒颗粒清晰而细胞扩增出的纯化后病毒颗粒背景较为杂乱,实验仍需进行下一步,在分辨率更高的冷冻电子显微镜下细致观察两者的不同以进一步分析不同结构引起的差异。更多还原

【Abstract】 Respiratory virus is the main virus that infects the human respiratory system.This disease is widely prevalent in the population,causing cough,headache,fatigue,severe lung disease and other related symptoms,and even death.Therefore,it has attracted widespread attention worldwide.Human respiratory syncytial virus（HRSV）in the elderly and infants and young children with lower immunity,such as the crowd spread widely,causing respiratory infections,causing diseases such as pneumonia and bronchitis,brought huge losses,two kinds of structure of RSV protein F and G protein mediated the combination of virus and host cell,the virus can enter the cell.RSV F protein also has strong immunogenicity,so RSV F protein is considered as an important target in vaccine and drug research and development.The HA protein in influenza viruses,which have caused several pandemics and killed tens of millions of people around the world,mediates the fusion of the virus with the cell membrane,which allows the virus to enter the cell to replicate.RSV virus genome encoding 11 kinds of proteins,which encode the F protein is now popular target protein,which interact with a variety of a variety of proteins in the human body,the original form of the F protein is known as the F₀,F₀protein subunits was cut up to produce F₂,and F₁fusion as a unit,the unit three together form a trimer,At this point,the F protein is functioning normally.In this experiment,F protein gene was synthesized for point mutation and secreted and expressed in HEK293E and Expi293F.After concentration by ultrafiltration,it was carried out affinity chromatography with Ni-NTA.Stable and uniform proteins were obtained by molecular sieve Superose ^TM6 Increase 10/300 GL gel chromatography for negative staining by electron microscopy.Subsequently,proteins that interact with them could be prepared by using similar preparation methods and then combined,and the results were observed under frozen electron microscopy for further analysis.The results of this study lay a solid foundation for the subsequent research on antigenicity and immunogenicity of virus F protein.Most flu vaccines in chicken eggs for matrix as a preparation for production,however,chicken embryos with quantitative restrictions and pollution in the process of production is likely to occur,so the cells as matrix of flu vaccine also gradually began to develop,the MDCK cells is considered sensitive cells of the influenza virus,there are for substrates by MDCK cells of flu vaccine.In different substrates,the HA protein of influenza virus may be different in structure and quantity,and HA protein plays an important role in the infection process of influenza virus.Whether different structure of HA protein will affect the effect of vaccine is still needed to be further explored.In this experiment,we respectively in chicken embryos and MDCK cells proliferation H3N2virus,and the filtered disease venom sucrose density gradient centrifugation and purification,again after PCR detection of the viral nucleic acid in negative dyeing observation by electron microscope,found that by chicken embryos amplification of viral particles and by MDCK cells proliferation of viral particles size and the shape is slightly different,The purified virus particles amplified from chicken embryos were clear,while the purified virus particles amplified from cells had a more chaotic background.The experiment still needed to proceed to the next step,and the differences between the two were carefully observed under cryonic electron microscopy with higher resolution to further analyze the differences caused by different structures.更多还原