本文選題：肺炎多糖結(jié)合蛋白載體疫苗 + 點(diǎn)擊化學(xué)��； 參考：《福建農(nóng)林大學(xué)》2013年碩士論文

【摘要】：肺炎鏈球菌是一種常見(jiàn)的病原微生物，其侵入人體可引起肺炎球菌綜合癥（IPD）的發(fā)生，其主要病癥包括：敗血癥、腦膜炎、肺炎及中耳炎等。肺炎莢膜多糖是一種天然抗原大分子，被廣泛用于疫苗生產(chǎn)，但由于其不能夠激起T細(xì)胞免疫，單獨(dú)使用，對(duì)于免疫力低下人群，如：老人，，化療患者及2歲以下嬰兒來(lái)講效果并不明顯。因此，經(jīng)過(guò)研究，2000年在美國(guó)上市的7價(jià)肺炎莢膜多糖結(jié)合蛋白載體疫苗在一定程度上解決了這一問(wèn)題。 然而傳統(tǒng)方法制備肺炎多糖結(jié)合疫苗的工藝中存在諸多問(wèn)題，只要總結(jié)為兩點(diǎn)：（1）由于多糖與載體蛋白均具有較大的分子量，因此所生成的肺炎多糖結(jié)合疫苗不可避免的存在空間屏蔽效應(yīng)，可能導(dǎo)致抗原表位被遮蔽，無(wú)法更好的發(fā)揮出肺炎多糖結(jié)合疫苗的免疫學(xué)作用。（2）從工藝方面講，傳統(tǒng)的生產(chǎn)方式周期較長(zhǎng)，往往需要5-6天甚至更長(zhǎng)，極大的增加了生產(chǎn)的成本。本次實(shí)驗(yàn)的目的旨在利用“點(diǎn)擊化學(xué)”在多糖與TT蛋白交聯(lián)過(guò)程中引入長(zhǎng)鏈Linker做為連接橋，從而使大分子間的距離增大，以減少空間屏蔽作用對(duì)肺炎多糖結(jié)合疫苗免疫學(xué)性質(zhì)的影響，并縮短生產(chǎn)周期。 一、制備偶聯(lián)物。采用AKTA蛋白純化系統(tǒng)與制備型Superdex200凝膠過(guò)濾柱(2.6cm×70cm)純化破傷風(fēng)類(lèi)毒素蛋白，純度達(dá)到98%，可用于實(shí)驗(yàn)。采用“點(diǎn)擊化學(xué)”法進(jìn)行偶聯(lián)，可有效的將生產(chǎn)周期縮短至2天。采用AKTA蛋白純化系統(tǒng)與制備型Superdex200凝膠過(guò)濾柱(2.6cm×70cm)制備結(jié)合疫苗。 二、對(duì)偶聯(lián)產(chǎn)物進(jìn)行結(jié)構(gòu)和免疫學(xué)性質(zhì)的表征。首先利用內(nèi)源熒光光譜、動(dòng)態(tài)光散射技術(shù)，圓二色技術(shù)，核磁共振技術(shù)及酶解技術(shù)對(duì)2種疫苗的結(jié)構(gòu)進(jìn)行表征。與TT相比，發(fā)現(xiàn)結(jié)合疫苗的內(nèi)源熒光出現(xiàn)明顯的下降，分析認(rèn)為結(jié)合疫苗中“點(diǎn)擊化學(xué)”反應(yīng)中生成的五元雜環(huán)起到熒光猝滅作用；偶聯(lián)后流體力學(xué)半徑都明顯增加；圓二色光譜與核磁共振結(jié)果顯示，偶聯(lián)后TT蛋白與多糖結(jié)構(gòu)均未發(fā)生明顯改變；對(duì)包括兩種疫苗及TT蛋白在內(nèi)的3種樣品進(jìn)行酶解分子發(fā)現(xiàn)，長(zhǎng)鏈連接橋可有效的降低空間位阻對(duì)多糖抗原的影響。另外，免疫學(xué)性質(zhì)檢測(cè)顯示，長(zhǎng)鏈連接橋可提高有效小鼠抗肺炎多糖IgM與IgG的抗體滴度，對(duì)所產(chǎn)生的IgG抗體亞型進(jìn)行分析發(fā)現(xiàn)，長(zhǎng)鏈組中IgG2a在總IgG中所占比例高于短鏈組，分子認(rèn)為長(zhǎng)鏈組疫苗可有效的激起小鼠的T細(xì)胞免疫反應(yīng)。
[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.
【學(xué)位授予單位】：福建農(nóng)林大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2013
【分類(lèi)號(hào)】：R563.1;R392.1

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