经MHC-Ⅱ通路的粉尘螨Ⅰ类变应原ProDer f 1疫苗设计及其免疫治疗效果评价
发布时间：2019-02-19 14:45
【摘要】：目的：构建编码DCP-IhC-ProDer f1嵌合基因的原核表达载体pET28a(+)-DCP-IhC-ProDer f1并进行大规模表达纯化，探讨嵌合变应原DCP-IhC-ProDer f1的特异性免疫治疗效果。 方法：⑴合成特异性DC细胞结合肽（DCP）和MHC-Ⅱ通路中的恒定链（invariant chain, Ii）的1-110个氨基酸残基的基因片段，用分子生物学方法分别构建嵌合基因DCP-ProDer f1和DCP-IhC-ProDer f1，并插入到原核表达载体pET28a(+)中，形成重组原核表达载体pET28a(+)-DCP-ProDer f1和pET28a(+)-DCP-IhC-ProDer f1，限制性内切酶和测序验证；⑵将重组原核表达载体转入大肠杆菌E.coli BL21（DE3）菌株，用IPTG小剂量诱导DCP-ProDerf1和DCP-IhC-ProDer f1的表达，优化条件后进行大规模表达、纯化；⑶构建ProDer f1变应原原致敏的小鼠哮喘模型，分别以纯化的嵌合变应原DCP-ProDer f1和DCP-IhC-ProDer f1为疫苗，对哮喘小鼠模型进行特异性免疫治疗后，ELISA法检测支气管肺泡灌洗液和脾细胞培养上清中IFN-γ、IL-4、IL-10和IL-17的水平以及血清中变应原特异性IgE、IgG1和IgG2a抗体水平。同时检测肺组织病理变化。 结果:⑴限制性内切酶双酶切和测序结果证实，成功构建了pET28a(+)-DCP-ProDer f1和pET28a(+)-DCP-IhC-ProDer f1原核表达载体；⑵IPTG诱导含pET28a(+)-DCP-ProDer f1和pET28a(+)-DCP-IhC-ProDer f1的E. coli BL21（DE3）菌株后，经SDS-PAGE和Western blot分析表明，成功诱导出DCP-ProDer f1和DCP-IhC-ProDer f1嵌合变应原；同时完成了大规模纯化；⑶用DCP-ProDer f1和DCP-IhC-ProDer f1嵌合变应原对ProDer f1致敏的小鼠哮喘模型进行特异性免疫治疗后，肺组织切片观察显示：各治疗组变态反应性炎症较哮喘模型组明显减轻，表现为炎性细胞浸润减少。ELISA法检测结果表明：各免疫治疗组中血清抗原特异性IgE和IgG1抗体明显低于哮喘组（p0.05），而IgG2a抗体水平显著升高（p0.05）；BALF和脾细胞培养上清中的IL-4和IL-17水平均低于哮喘组（p0.05）；而BALF和脾细胞培养分泌IFN-γ和IL-10含量均高于哮喘组（p0.05）。对上述指标的分析发现，，DCP-IhC-ProDer f1的免疫治疗效果优于DCP-ProDer f1及ProDer f1（p0.05），而DCP-ProDer f1与ProDer f1组之间的疗效相近（p0.05）。 结论:成功构建了原核表达载体pET28a(+)-DCP-ProDer f1、pET28a(+)-DCP-IhC-ProDer f1并进行了大规模纯化；表达的嵌合蛋白对小鼠哮喘模型进行特异性免疫治疗后可有效改善小鼠变态反应性气道及肺部炎症。
[Abstract]:Objective: To construct a prokaryotic expression vector pET28a (+)-DCP-IhC-ProDer f1 encoding a DCP-IhC-ProDer f1 chimeric gene and to carry out large-scale expression and purification to study the specific immunotherapeutic effect of the chimeric allergen DCP-IhC-ProDer f1. Methods: The gene fragments of 1 to 110 amino acid residues of the constant chain (Ii) in the specific DC cell binding peptide (DCP) and the MHC-II pathway were synthesized, and the chimeric genes DCP-ProDer f1 and DCP-IhC-ProDer f1 were constructed by molecular biological methods, and inserted into the prokaryotic expression vector pET28a (+). The recombinant prokaryotic expression vector pET28a (+)-DCP-ProDer f1 and pET28a (+)-DCP-IhC-ProDer f1, restriction-restriction enzyme and sequencing verification were formed, and the recombinant prokaryotic expression vector was transferred to E. coli BL21 (DE3) strain, and the expression of DCP-ProDerf1 and DCP-IhC-ProDer f1 was induced with low-dose IPTG. The mouse asthma model of ProDer f1 allergen was constructed, and the purified chimeric allergen DCP-ProDer f1 and DCP-IhC-ProDer f1 were used as the vaccine, and after the specific immunotherapy of the asthma mouse model, the IFN-1 and IL-2 in the supernatant of the bronchoalveolar lavage fluid and the spleen cell culture were detected by ELISA. 4. Levels of IL-10 and IL-17 as well as the allergen specific IgE, IgG1 and IgG2a antibody water in the serum The pathological changes of the lung tissue were detected at the same time. The results showed that the expression vector of E. coli BL21 (DE3) containing pET28a (+)-DCP-ProDer f1 and pET28a (+)-DCP-IhC-ProDer f1 was successfully constructed. After the E. coli BL21 (DE3) strain containing pET28a (+)-DCP-ProDer f1 and pET28a (+)-DCP-IhC-ProDer f1 was induced by IPTG, the expression of DCP-ProDer f1 and DCP-IhC-ProDer f1 was successfully induced. Allergens were simultaneously completed; after specific immunotherapy with DCP-ProDer f1 and DCP-IhC-ProDer f1 chimeric allergen to the mouse asthma model of ProDer f1, the lung tissue sections showed that the allergic inflammation in each treatment group was significantly reduced compared with that of the asthma model group and expressed as an inflammatory cell. The results of ELISA showed that the serum antigen-specific IgE and IgG1 antibody in each immunotherapy group were significantly lower than that of the asthma group (p0.05), while the level of the IgG2a antibody was significantly higher (p0.05), and the level of IL-4 and IL-17 in the supernatant of BALF and spleen cell culture was lower than that of the asthma group (p0. The content of IFN-1 and IL-10 in BALF and spleen cells was higher than that of asthma group (p0). The results showed that the therapeutic effect of DCP-IhC-ProDer f1 was better than that of DCP-ProDer f1 and ProDer f1 (p0.05), while the efficacy of DCP-ProDer f1 and ProDer f1 was similar (p0. Conclusion: The prokaryotic expression vector pET28a (+)-DCP-ProDer f1, pET28a (+)-DCP-IhC-ProDer f1 was successfully constructed and a large-scale purification was carried out. The expressed chimeric protein can effectively improve the allergic gas channel of the mouse after the specific immunotherapy of the mouse asthma model.
【学位授予单位】：皖南医学院
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R392
[Abstract]:Objective: To construct a prokaryotic expression vector pET28a (+)-DCP-IhC-ProDer f1 encoding a DCP-IhC-ProDer f1 chimeric gene and to carry out large-scale expression and purification to study the specific immunotherapeutic effect of the chimeric allergen DCP-IhC-ProDer f1. Methods: The gene fragments of 1 to 110 amino acid residues of the constant chain (Ii) in the specific DC cell binding peptide (DCP) and the MHC-II pathway were synthesized, and the chimeric genes DCP-ProDer f1 and DCP-IhC-ProDer f1 were constructed by molecular biological methods, and inserted into the prokaryotic expression vector pET28a (+). The recombinant prokaryotic expression vector pET28a (+)-DCP-ProDer f1 and pET28a (+)-DCP-IhC-ProDer f1, restriction-restriction enzyme and sequencing verification were formed, and the recombinant prokaryotic expression vector was transferred to E. coli BL21 (DE3) strain, and the expression of DCP-ProDerf1 and DCP-IhC-ProDer f1 was induced with low-dose IPTG. The mouse asthma model of ProDer f1 allergen was constructed, and the purified chimeric allergen DCP-ProDer f1 and DCP-IhC-ProDer f1 were used as the vaccine, and after the specific immunotherapy of the asthma mouse model, the IFN-1 and IL-2 in the supernatant of the bronchoalveolar lavage fluid and the spleen cell culture were detected by ELISA. 4. Levels of IL-10 and IL-17 as well as the allergen specific IgE, IgG1 and IgG2a antibody water in the serum The pathological changes of the lung tissue were detected at the same time. The results showed that the expression vector of E. coli BL21 (DE3) containing pET28a (+)-DCP-ProDer f1 and pET28a (+)-DCP-IhC-ProDer f1 was successfully constructed. After the E. coli BL21 (DE3) strain containing pET28a (+)-DCP-ProDer f1 and pET28a (+)-DCP-IhC-ProDer f1 was induced by IPTG, the expression of DCP-ProDer f1 and DCP-IhC-ProDer f1 was successfully induced. Allergens were simultaneously completed; after specific immunotherapy with DCP-ProDer f1 and DCP-IhC-ProDer f1 chimeric allergen to the mouse asthma model of ProDer f1, the lung tissue sections showed that the allergic inflammation in each treatment group was significantly reduced compared with that of the asthma model group and expressed as an inflammatory cell. The results of ELISA showed that the serum antigen-specific IgE and IgG1 antibody in each immunotherapy group were significantly lower than that of the asthma group (p0.05), while the level of the IgG2a antibody was significantly higher (p0.05), and the level of IL-4 and IL-17 in the supernatant of BALF and spleen cell culture was lower than that of the asthma group (p0. The content of IFN-1 and IL-10 in BALF and spleen cells was higher than that of asthma group (p0). The results showed that the therapeutic effect of DCP-IhC-ProDer f1 was better than that of DCP-ProDer f1 and ProDer f1 (p0.05), while the efficacy of DCP-ProDer f1 and ProDer f1 was similar (p0. Conclusion: The prokaryotic expression vector pET28a (+)-DCP-ProDer f1, pET28a (+)-DCP-IhC-ProDer f1 was successfully constructed and a large-scale purification was carried out. The expressed chimeric protein can effectively improve the allergic gas channel of the mouse after the specific immunotherapy of the mouse asthma model.
【学位授予单位】：皖南医学院
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R392
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