新型肺炎多糖结合蛋白载体疫苗的制备及表征

发布时间：2018-04-30 22:06

本文选题：肺炎多糖结合蛋白载体疫苗 + 点击化学　；参考：《福建农林大学》2013年硕士论文

【摘要】：肺炎链球菌是一种常见的病原微生物，其侵入人体可引起肺炎球菌综合症（IPD）的发生，其主要病症包括：败血症、脑膜炎、肺炎及中耳炎等。肺炎荚膜多糖是一种天然抗原大分子，被广泛用于疫苗生产，但由于其不能够激起T细胞免疫，单独使用，对于免疫力低下人群，如：老人，，化疗患者及2岁以下婴儿来讲效果并不明显。因此，经过研究，2000年在美国上市的7价肺炎荚膜多糖结合蛋白载体疫苗在一定程度上解决了这一问题。然而传统方法制备肺炎多糖结合疫苗的工艺中存在诸多问题，只要总结为两点：（1）由于多糖与载体蛋白均具有较大的分子量，因此所生成的肺炎多糖结合疫苗不可避免的存在空间屏蔽效应，可能导致抗原表位被遮蔽，无法更好的发挥出肺炎多糖结合疫苗的免疫学作用。（2）从工艺方面讲，传统的生产方式周期较长，往往需要5-6天甚至更长，极大的增加了生产的成本。本次实验的目的旨在利用“点击化学”在多糖与TT蛋白交联过程中引入长链Linker做为连接桥，从而使大分子间的距离增大，以减少空间屏蔽作用对肺炎多糖结合疫苗免疫学性质的影响，并缩短生产周期。一、制备偶联物。采用AKTA蛋白纯化系统与制备型Superdex200凝胶过滤柱(2.6cm×70cm)纯化破伤风类毒素蛋白，纯度达到98%，可用于实验。采用“点击化学”法进行偶联，可有效的将生产周期缩短至2天。采用AKTA蛋白纯化系统与制备型Superdex200凝胶过滤柱(2.6cm×70cm)制备结合疫苗。二、对偶联产物进行结构和免疫学性质的表征。首先利用内源荧光光谱、动态光散射技术，圆二色技术，核磁共振技术及酶解技术对2种疫苗的结构进行表征。与TT相比，发现结合疫苗的内源荧光出现明显的下降，分析认为结合疫苗中“点击化学”反应中生成的五元杂环起到荧光猝灭作用；偶联后流体力学半径都明显增加；圆二色光谱与核磁共振结果显示，偶联后TT蛋白与多糖结构均未发生明显改变；对包括两种疫苗及TT蛋白在内的3种样品进行酶解分子发现，长链连接桥可有效的降低空间位阻对多糖抗原的影响。另外，免疫学性质检测显示，长链连接桥可提高有效小鼠抗肺炎多糖IgM与IgG的抗体滴度，对所产生的IgG抗体亚型进行分析发现，长链组中IgG2a在总IgG中所占比例高于短链组，分子认为长链组疫苗可有效的激起小鼠的T细胞免疫反应。
[Abstract]:Streptococcus pneumoniae (Streptococcus pneumoniae) is a common pathogenic microorganism, which invades the human body to cause the occurrence of pneumococcal syndrome (IPD). Its main symptoms include septicemia, meningitis, pneumonia and otitis media. Pneumonia capsule polysaccharide is a kind of natural antigen macromolecule, which is widely used in vaccine production, but because it can not arouse T cell immunity, it can be used alone for people with low immunity, such as the elderly, Chemotherapy patients and children under 2 years of age were not effective. Therefore, the 7 valent pneumonia capsule polysaccharide binding protein vector vaccine, which was launched in the United States in 2000, has solved this problem to a certain extent. However, there are many problems in the process of preparing pneumonic polysaccharide conjugated vaccine by traditional method, as long as it is summed up as two points: 1) because the polysaccharide and carrier protein have higher molecular weight, Therefore, the resulting pneumonic polysaccharide binding vaccine inevitably has a space shielding effect, which may result in the antigen epitopes being obscured and unable to play a better role in the immunology of the pneumonic polysaccharide bound vaccine. The traditional production cycle is longer, often 5-6 days or longer, greatly increasing the cost of production. The aim of this study was to use "click-chemistry" to introduce long-stranded Linker into the crosslinking process between polysaccharides and TT proteins, thus increasing the distance between macromolecules. In order to reduce the effect of space shielding on the immunological properties of pneumonic polysaccharide-bound vaccine and shorten the production cycle. First, the conjugate was prepared. Tetanus toxoid protein was purified by AKTA protein purification system and prepared Superdex200 gel filtration column (2.6cm 脳 70cm). The purity of tetanus toxoid protein was 98. It can be used in experiments. By using click-chemistry method, the production cycle can be effectively shortened to 2 days. The conjugated vaccine was prepared by using AKTA protein purification system and prepared Superdex200 gel filtration column of 2.6 cm 脳 70 cm. Second, the structure and immunological properties of the coupling products were characterized. Firstly, the structures of the two vaccines were characterized by endogenous fluorescence spectroscopy, dynamic light scattering, circular dichroism, nuclear magnetic resonance and enzymatic hydrolysis. Compared with TT, it was found that the endogenous fluorescence of the conjugated vaccine decreased obviously, and the fluorescence quenching effect of the five-member heterocycle produced in the click-chemical reaction was found, and the hydrodynamic radius of the conjugated vaccine increased obviously after coupling. The results of circular dichroism and nuclear magnetic resonance (NMR) showed that the structure of TT protein and polysaccharide did not change obviously after coupling, and three kinds of samples, including two vaccines and TT protein, were detected by enzymolysis. Long chain bridging can effectively reduce the effect of steric resistance on polysaccharide antigen. In addition, the immunological properties showed that the antibody titers of IgM and IgG could be increased by long chain junction bridge. The results showed that the proportion of IgG2a in total IgG in the long chain group was higher than that in the short chain group, and the subtype of IgG antibody in the long chain group was higher than that in the short chain group. It is believed that the long chain vaccine can effectively stimulate T cell immune response in mice.
【学位授予单位】：福建农林大学
【学位级别】：硕士
【学位授予年份】：2013
【分类号】：R563.1;R392.1

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