【摘要】：DNA初免-蛋白質(zhì)加強的免疫策略已經(jīng)廣泛應(yīng)用以增強這兩種疫苗的免疫原性,但是這種免疫增效的機制目前還不清楚。我們以pVAX1質(zhì)粒為載體構(gòu)建了表達乙肝表面抗原的真核表達質(zhì)粒pVAX-S,用pVAX-S和重組乙肝表面抗原以C57BL/6小鼠為實驗動物實施了DDD、DDS、SSS和SSD4種不同的免疫策略。在第一次免疫后第3周、7周、11周、15周、19周和23周用酶聯(lián)免疫吸附法檢測了這些小鼠的總抗體滴度,第11周用混合淋巴細胞反應(yīng)檢測了不同免疫組小鼠的T細胞免疫記憶強度和樹突狀細胞刺激T細胞增殖的能力,第23周用RocheAssay Medium分光法按效應(yīng)細胞/靶細胞1:50的比例檢測了CTL活性。我們觀察到DDS組小鼠擁有最強的免疫記憶,無論抗體滴度、CTL活性、T細胞免疫記憶強度還是樹突狀細胞激活T細胞的能力都比其它免疫組以及空白對照組的小鼠更強。我們進一步進行了比較血清蛋白質(zhì)組學(xué)的研究,以期能闡明這種免疫增效作用的機制。以非免疫組小鼠作參照膠,我們發(fā)現(xiàn)DDS組小鼠有5個蛋白上調(diào)、17個蛋白下調(diào);而SSS組小鼠則有7個蛋白上調(diào)、10個蛋白下調(diào)。在DDS組中唯一出現(xiàn)的3個蛋白質(zhì)periplakin、F-box protein 30和calpain的生理功能都和免疫反應(yīng)有一定的關(guān)系。這3個蛋白可以作為潛在的成功免疫的新型分子標記物,也可能成為疫苗作用機制的新研究目標。在我們的一個在研項目中,我們用豬帶絳蟲囊尾蚴抗原膜聯(lián)蛋白B1 DNA疫苗和蛋白質(zhì)疫苗對小鼠進行免疫后分析其血清蛋白質(zhì)組,初步得到6個特異性表達蛋白的質(zhì)譜結(jié)果,這6個蛋白與乙肝表面抗原DDS/SSS免疫組小鼠血清中鑒定的6個特異性表達蛋白無一相同,說明這3個從DDS免疫組中發(fā)現(xiàn)的特異性表達蛋白具有抗原特異性。
[Abstract]:DNA immune-protein enhanced immunization strategy has been widely used to enhance the immunogenicity of the two vaccines, but the mechanism of this immune synergism is not clear. The eukaryotic expression plasmid pVAX-S, expressing hepatitis B surface antigen was constructed by using pVAX1 plasmid as vector. The different immunological strategies of DDD,DDS,SSS and SSD4 were carried out by using pVAX-S and C57BL/6 mice as experimental animals. The total antibody titers of the mice were detected by enzyme-linked immunosorbent assay (Elisa) at week 3, week 7, week 11, week 15, week 19 and week 23 after the first immunization. At the 11th week, the T cell immune memory intensity and the ability of dendritic cells to stimulate T cell proliferation were measured by mixed lymphocyte reaction in mice of different immune groups. The activity of CTL was measured by RocheAssay Medium spectrophotometry at 1:50 of effector cell / target cell at the 23rd week. We observed that DDS mice had the strongest immune memory, whether antibody titer, CTL activity, T cell immune memory intensity or the ability of dendritic cells to activate T cells were stronger than those of other immune groups and blank control groups. We further studied the comparison of serum proteomics in order to elucidate the mechanism of this immune synergism. Using non-immunized mice as reference glue, we found that 5 proteins were up-regulated and 17 proteins down-regulated in DDS group, while 7 proteins were up-regulated and 10 proteins down-regulated in SSS group. The physiological functions of periplakin,F-box protein 30 and calpain, the only three proteins in DDS group, were related to the immune response to some extent. These three proteins can be used as potential novel molecular markers for successful immunization, and may also become new research targets for the mechanism of vaccine action. In one of our research projects, we immunized mice with cysticercus cellulosae antigen integrin B1 DNA vaccine and protein vaccine and analyzed their serum proteome, and obtained the mass spectrometry results of six specific expressed proteins. These six proteins were not the same as those identified in the serum of mice immunized with hepatitis B surface antigen (DDS/SSS), indicating that the three specific expressed proteins from DDS immunized group had antigen-specificity.
【學(xué)位授予單位】：第二軍醫(yī)大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2006
【分類號】：R392

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