【摘要】：目的：構(gòu)建編碼DCP-IhC-ProDer f1嵌合基因的原核表達(dá)載體pET28a(+)-DCP-IhC-ProDer f1并進(jìn)行大規(guī)模表達(dá)純化，探討嵌合變應(yīng)原DCP-IhC-ProDer f1的特異性免疫治療效果。 方法：⑴合成特異性DC細(xì)胞結(jié)合肽（DCP）和MHC-Ⅱ通路中的恒定鏈（invariant chain, Ii）的1-110個(gè)氨基酸殘基的基因片段，用分子生物學(xué)方法分別構(gòu)建嵌合基因DCP-ProDer f1和DCP-IhC-ProDer f1，并插入到原核表達(dá)載體pET28a(+)中，形成重組原核表達(dá)載體pET28a(+)-DCP-ProDer f1和pET28a(+)-DCP-IhC-ProDer f1，限制性內(nèi)切酶和測(cè)序驗(yàn)證；⑵將重組原核表達(dá)載體轉(zhuǎn)入大腸桿菌E.coli BL21（DE3）菌株，用IPTG小劑量誘導(dǎo)DCP-ProDerf1和DCP-IhC-ProDer f1的表達(dá)，優(yōu)化條件后進(jìn)行大規(guī)模表達(dá)、純化；⑶構(gòu)建ProDer f1變應(yīng)原原致敏的小鼠哮喘模型，分別以純化的嵌合變應(yīng)原DCP-ProDer f1和DCP-IhC-ProDer f1為疫苗，對(duì)哮喘小鼠模型進(jìn)行特異性免疫治療后，ELISA法檢測(cè)支氣管肺泡灌洗液和脾細(xì)胞培養(yǎng)上清中IFN-γ、IL-4、IL-10和IL-17的水平以及血清中變應(yīng)原特異性IgE、IgG1和IgG2a抗體水平。同時(shí)檢測(cè)肺組織病理變化。 結(jié)果:⑴限制性內(nèi)切酶雙酶切和測(cè)序結(jié)果證實(shí)，成功構(gòu)建了pET28a(+)-DCP-ProDer f1和pET28a(+)-DCP-IhC-ProDer f1原核表達(dá)載體；⑵IPTG誘導(dǎo)含pET28a(+)-DCP-ProDer f1和pET28a(+)-DCP-IhC-ProDer f1的E. coli BL21（DE3）菌株后，經(jīng)SDS-PAGE和Western blot分析表明，成功誘導(dǎo)出DCP-ProDer f1和DCP-IhC-ProDer f1嵌合變應(yīng)原；同時(shí)完成了大規(guī)模純化；⑶用DCP-ProDer f1和DCP-IhC-ProDer f1嵌合變應(yīng)原對(duì)ProDer f1致敏的小鼠哮喘模型進(jìn)行特異性免疫治療后，肺組織切片觀察顯示：各治療組變態(tài)反應(yīng)性炎癥較哮喘模型組明顯減輕，表現(xiàn)為炎性細(xì)胞浸潤(rùn)減少。ELISA法檢測(cè)結(jié)果表明：各免疫治療組中血清抗原特異性IgE和IgG1抗體明顯低于哮喘組（p0.05），而IgG2a抗體水平顯著升高（p0.05）；BALF和脾細(xì)胞培養(yǎng)上清中的IL-4和IL-17水平均低于哮喘組（p0.05）；而B(niǎo)ALF和脾細(xì)胞培養(yǎng)分泌IFN-γ和IL-10含量均高于哮喘組（p0.05）。對(duì)上述指標(biāo)的分析發(fā)現(xiàn)，，DCP-IhC-ProDer f1的免疫治療效果優(yōu)于DCP-ProDer f1及ProDer f1（p0.05），而DCP-ProDer f1與ProDer f1組之間的療效相近（p0.05）。 結(jié)論:成功構(gòu)建了原核表達(dá)載體pET28a(+)-DCP-ProDer f1、pET28a(+)-DCP-IhC-ProDer f1并進(jìn)行了大規(guī)模純化；表達(dá)的嵌合蛋白對(duì)小鼠哮喘模型進(jìn)行特異性免疫治療后可有效改善小鼠變態(tài)反應(yīng)性氣道及肺部炎癥。
[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.
【學(xué)位授予單位】：皖南醫(yī)學(xué)院
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2014
【分類號(hào)】：R392

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