結(jié)核亞單位疫苗Mtb8.4-HspX和HspX-Mtb8.4表達(dá)純化及免疫保護(hù)效果研究
發(fā)布時(shí)間：2018-12-11 04:31
【摘要】：目的：結(jié)核病是困擾人類健康的重要疾病,研制新型有效、安全的結(jié)核疫苗成為國內(nèi)外學(xué)者共同關(guān)注的一個(gè)重要課題。結(jié)核亞單位疫苗成為最有發(fā)展前景的新型疫苗之一,同時(shí)也存在著很多影響亞單位疫苗免疫保護(hù)效率的因素,如融合蛋白的組成順序與接種劑量。研究亞單位疫苗的蛋白組成順序和接種劑量與免疫保護(hù)效率的關(guān)系,對(duì)合理設(shè)計(jì)疫苗和優(yōu)化免疫策略有重要的意義。 方法：有兩個(gè)結(jié)核疫苗候選抗原被用于我們的實(shí)驗(yàn)研究中,分別為Mtb8.4和HspX。利用生物工程的原理和方法,我們成功制備了兩個(gè)融合蛋白Mtb8.4-HspX和HspX-Mtb8.4,分別簡稱為8.4H和H8.4。用8.4H和H8.4體外刺激結(jié)核病人及結(jié)核分支桿菌潛伏感染人群的外周血淋巴細(xì)胞,ELISPOT方法檢測各組IFN-Y分泌的水平。將佐劑DDA和poly(I:C)與助溶劑明膠按照一定比例混勻制成復(fù)合佐劑,簡稱DPG。復(fù)合佐劑與融合蛋白8.4H和H8.4混合制成均一穩(wěn)定的亞單位疫苗,分別簡稱8.4H-DPG和H8.4-DPG,而后免疫C57BL/6小鼠,進(jìn)行免疫檢測和攻毒實(shí)驗(yàn),比較兩組疫苗的免疫保護(hù)效率和組織病理學(xué)差異。鑒于明膠對(duì)免疫保護(hù)效率的負(fù)影響和佐劑DDA與poly(I:C)的混合問題,我們僅用佐劑DDA分別與蛋白8.4H和H8.4制成亞單位疫苗,分別稱為8.4H-DDA和H8.4-DDA,而后免疫C57BL/6小鼠,ELISA方法檢測各疫苗組Thl型的細(xì)胞因子IFN-γ、TNF-a和IL-2和Th2型細(xì)胞因子IL-4及IgG的水平,并分別比較8.4H-DDA組和H8.4-DDA組及8.4H-DDA高劑量組和低劑量組小鼠細(xì)胞免疫和體液免疫應(yīng)答水平。 結(jié)果：融合蛋白8.4H和H8.4均能夠刺激人PBMC分泌高水平的IFN-Y,且蛋白H8.4的免疫原性較8.4H強(qiáng)；對(duì)比8.4H-DPG和H8.4-DPG免疫保護(hù)效率的差異,發(fā)現(xiàn)H8.4-DPG顯示出更好的免疫保護(hù)效果；比較H8.4-DDA組小鼠和8.4H-DDA組小鼠體液免疫應(yīng)答水平,發(fā)現(xiàn)H8.4-DDA組小鼠產(chǎn)生IgG1的水平要顯著高于H8.4-DDA組,而IgG2c/IgG1的值明顯低于后者；對(duì)比8.4H-DDA組小鼠和H8.4-DDA組小鼠細(xì)胞免疫應(yīng)答水平,發(fā)現(xiàn)8.4H-DDA組小鼠產(chǎn)生IFN-γ、TNF-α的水平顯著高于H8.4-DDA(?)且,而兩組分泌IL-2的水平相近,且均未檢測到IL-4的分泌；比較8.4H-DDA高劑量組和低劑量組小鼠的體液免疫應(yīng)答水平,發(fā)現(xiàn)高劑量組小鼠產(chǎn)生IgG2b和IgG2c的水平顯著高于低劑量組,且IgG2c/IgG1的值明顯高于后者；比較8.4H-DDA高劑量組和低劑量組小鼠的細(xì)胞免疫應(yīng)答水平,發(fā)現(xiàn)高劑量組小鼠分泌IFN-γ、TNF-α的水平顯著高于低劑量組,而產(chǎn)生IL-2的水平剛好相反。 結(jié)論：實(shí)驗(yàn)室成功制備了融合蛋白Mtb8.4-HspX (8.4H)和HspX-Mtb8.4(H8.4)且能夠刺激結(jié)核病患者分泌較高水平的IFN-γ；結(jié)核亞單位疫苗Mtb8.4-HspX和HspX-Mtb8.4的蛋白組成順序和接種劑量與Th1型的免疫應(yīng)答面有著密不可分的關(guān)系,因此也影響著亞單位疫苗的免疫保護(hù)效果。
[Abstract]:Objective: tuberculosis is an important disease that puzzles human health. The development of new effective and safe TB vaccine has become an important issue that scholars at home and abroad pay attention to. Tuberculosis subunit vaccine has become one of the most promising new vaccines, and there are many factors that affect the immune protection efficiency of subunit vaccine, such as the sequence of fusion protein composition and the dose of vaccine. It is of great significance to study the relationship between the sequence of protein composition and the dose of subunit vaccine and the efficiency of immune protection for the rational design of vaccine and the optimization of immunization strategy. Methods: two TB vaccine candidate antigens were used in our experimental study, Mtb8.4 and HspX., respectively. Using the principles and methods of bioengineering, we successfully prepared two fusion proteins Mtb8.4-HspX and HspX-Mtb8.4, called 8.4H and H8.4respectively. Peripheral blood lymphocytes were stimulated with 8.4H and H8.4 in vitro in patients with tuberculosis and those with latent infection of Mycobacterium tuberculosis. The levels of IFN-Y secretion in each group were detected by ELISPOT method. Blend adjuvant DDA and poly (I: C) with gelatin in a certain proportion to form a composite adjuvant, DPG. for short The hybrid adjuvant was mixed with fusion protein 8.4H and H8.4 to prepare homogeneous and stable subunit vaccine, which was referred to as 8.4H-DPG and H8.4-DPGrespectively, then C57BL/6 mice were immunized. The immune protection efficiency and histopathological difference of the two groups were compared. In view of the negative effect of gelatin on immune protection efficiency and the mixing of adjuvant DDA and poly (I: C), we only use adjuvant DDA and protein 8.4H and H8.4 to prepare subunit vaccine, which is called 8.4H-DDA and H8.4-DDA. Then C57BL/6 mice were immunized. The levels of Thl type cytokines IFN- 緯, TNF-a, IL-2 and Th2 type IL-4 and IgG were detected by ELISA method in each vaccine group. The cellular and humoral immune responses of 8.4H-DDA group, H8.4-DDA group, high dose 8.4H-DDA group and low dose group were compared respectively. Results: the fusion proteins 8.4H and H8.4 could stimulate human PBMC to secrete high levels of IFN-Y, and the immunogenicity of H8.4 was stronger than that of 8.4H. Compared with the difference of immune protection efficiency between 8.4H-DPG and H8.4-DPG, it was found that H8.4-DPG showed better immune protection effect. By comparing the humoral immune response between H8.4-DDA group and 8.4H-DDA group, it was found that the level of IgG1 production in H8.4-DDA group was significantly higher than that in H8.4-DDA group, and the IgG2c/IgG1 level was significantly lower in H8.4-DDA group than in H8.4-DDA group. Compared with the level of cellular immune response in 8.4H-DDA group and H8.4-DDA group, it was found that the levels of IFN- 緯 and TNF- 偽 in 8.4H-DDA group were significantly higher than those in H8.4-DDA group. Moreover, the level of IL-2 secretion in the two groups was similar, and the secretion of IL-4 was not detected in the two groups. The humoral immune response level of mice in 8.4H-DDA high dose group and low dose group was compared. It was found that the level of IgG2b and IgG2c in high dose group was significantly higher than that in low dose group, and the value of IgG2c/IgG1 in high dose group was significantly higher than that in low dose group. The level of cellular immune response in 8.4H-DDA high dose group and low dose group was compared. It was found that the levels of IFN- 緯 and TNF- 偽 in high dose group were significantly higher than those in low dose group, but the level of IL-2 production was just the opposite. Conclusion: the fusion proteins Mtb8.4-HspX (8.4H) and HspX-Mtb8.4 (H8.4) have been successfully prepared in laboratory and can stimulate TB patients to secrete higher levels of IFN- 緯. The sequence of protein composition and dose of Mtb8.4-HspX and HspX-Mtb8.4 of TB subunit vaccine are closely related to the immune response surface of Th1 type, so it also affects the immune protection effect of subunit vaccine.
【學(xué)位授予單位】：蘭州大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2012
【分類號(hào)】：R392
本文編號(hào)：2371874
[Abstract]:Objective: tuberculosis is an important disease that puzzles human health. The development of new effective and safe TB vaccine has become an important issue that scholars at home and abroad pay attention to. Tuberculosis subunit vaccine has become one of the most promising new vaccines, and there are many factors that affect the immune protection efficiency of subunit vaccine, such as the sequence of fusion protein composition and the dose of vaccine. It is of great significance to study the relationship between the sequence of protein composition and the dose of subunit vaccine and the efficiency of immune protection for the rational design of vaccine and the optimization of immunization strategy. Methods: two TB vaccine candidate antigens were used in our experimental study, Mtb8.4 and HspX., respectively. Using the principles and methods of bioengineering, we successfully prepared two fusion proteins Mtb8.4-HspX and HspX-Mtb8.4, called 8.4H and H8.4respectively. Peripheral blood lymphocytes were stimulated with 8.4H and H8.4 in vitro in patients with tuberculosis and those with latent infection of Mycobacterium tuberculosis. The levels of IFN-Y secretion in each group were detected by ELISPOT method. Blend adjuvant DDA and poly (I: C) with gelatin in a certain proportion to form a composite adjuvant, DPG. for short The hybrid adjuvant was mixed with fusion protein 8.4H and H8.4 to prepare homogeneous and stable subunit vaccine, which was referred to as 8.4H-DPG and H8.4-DPGrespectively, then C57BL/6 mice were immunized. The immune protection efficiency and histopathological difference of the two groups were compared. In view of the negative effect of gelatin on immune protection efficiency and the mixing of adjuvant DDA and poly (I: C), we only use adjuvant DDA and protein 8.4H and H8.4 to prepare subunit vaccine, which is called 8.4H-DDA and H8.4-DDA. Then C57BL/6 mice were immunized. The levels of Thl type cytokines IFN- 緯, TNF-a, IL-2 and Th2 type IL-4 and IgG were detected by ELISA method in each vaccine group. The cellular and humoral immune responses of 8.4H-DDA group, H8.4-DDA group, high dose 8.4H-DDA group and low dose group were compared respectively. Results: the fusion proteins 8.4H and H8.4 could stimulate human PBMC to secrete high levels of IFN-Y, and the immunogenicity of H8.4 was stronger than that of 8.4H. Compared with the difference of immune protection efficiency between 8.4H-DPG and H8.4-DPG, it was found that H8.4-DPG showed better immune protection effect. By comparing the humoral immune response between H8.4-DDA group and 8.4H-DDA group, it was found that the level of IgG1 production in H8.4-DDA group was significantly higher than that in H8.4-DDA group, and the IgG2c/IgG1 level was significantly lower in H8.4-DDA group than in H8.4-DDA group. Compared with the level of cellular immune response in 8.4H-DDA group and H8.4-DDA group, it was found that the levels of IFN- 緯 and TNF- 偽 in 8.4H-DDA group were significantly higher than those in H8.4-DDA group. Moreover, the level of IL-2 secretion in the two groups was similar, and the secretion of IL-4 was not detected in the two groups. The humoral immune response level of mice in 8.4H-DDA high dose group and low dose group was compared. It was found that the level of IgG2b and IgG2c in high dose group was significantly higher than that in low dose group, and the value of IgG2c/IgG1 in high dose group was significantly higher than that in low dose group. The level of cellular immune response in 8.4H-DDA high dose group and low dose group was compared. It was found that the levels of IFN- 緯 and TNF- 偽 in high dose group were significantly higher than those in low dose group, but the level of IL-2 production was just the opposite. Conclusion: the fusion proteins Mtb8.4-HspX (8.4H) and HspX-Mtb8.4 (H8.4) have been successfully prepared in laboratory and can stimulate TB patients to secrete higher levels of IFN- 緯. The sequence of protein composition and dose of Mtb8.4-HspX and HspX-Mtb8.4 of TB subunit vaccine are closely related to the immune response surface of Th1 type, so it also affects the immune protection effect of subunit vaccine.
【學(xué)位授予單位】：蘭州大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2012
【分類號(hào)】：R392
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相關(guān)期刊論文 前3條
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