本文選題：Flt3L + 分子佐劑�。� 參考：《暨南大學》2009年碩士論文

【摘要】： 目的: 腫瘤和慢性病毒感染是當前人類面臨的重大挑戰(zhàn),而免疫治療是針對腫瘤和慢性病毒感染的有前途的新策略。但是,現(xiàn)有的臨床研究表明包括治療性疫苗在內(nèi)的免疫治療策略的療效甚微,因此,尋找如何有效增強免疫治療效果的新策略已成為當前的研究熱點。本研究以LCMV肽疫苗為模型,探討Ftl3L分子佐劑對KAV肽疫苗的免疫應(yīng)答的影響,并初步分析其作用機制,為Flt3L分子佐劑的臨床研究提供參考。 方法: 通過對現(xiàn)有的Flt3L基因信息進行分析設(shè)計引物,RT-PCR法擴增并克隆其cDNA,進而分別構(gòu)建Flt3L原核表達質(zhì)粒、可溶型和全長Flt3L真核表達質(zhì)粒,測序驗證后,分別轉(zhuǎn)化大腸桿菌或利用FuGENE HD試劑轉(zhuǎn)染HeLa細胞進行表達,應(yīng)用SDS-PAGE和/或免疫印跡檢測其表達情況。然后,進行真核細胞轉(zhuǎn)染后,對含有目標蛋白的細胞培養(yǎng)上清進行生物學活性檢測�？缒ば虵lt3L真核表達載體作為分子佐劑,在小鼠中評價其對LCMV肽疫苗誘發(fā)的免疫應(yīng)答的影響,利用四聚體技術(shù)分析其對KAV特異性CD8~+T細胞頻率與表型的干預(yù)作用。 結(jié)果: 1、通過RT-PCR獲得與以往報道序列一致的Flt3L編碼區(qū)全長cDNA,并已遞交GenBank,登錄號為EU274583;然后,成功構(gòu)建了Flt3L的胞外域原核表達載體并測序正確,證實其能在大腸桿菌BL21(DE3)內(nèi)高效表達。 2、進而構(gòu)建可溶型Flt3L真核表達載體并測序驗證正確,然后在HeLa細胞進行表達,獲得48 h細胞培養(yǎng)上清。以小鼠骨髓細胞的增殖和甲基纖維素半固體培養(yǎng)集落培養(yǎng)實驗進行分析,顯示轉(zhuǎn)染可溶型Flt3L的細胞培養(yǎng)上清具有一定的生物活性,能夠促進GM-CSF引起的集落形成。 3、同時進一步構(gòu)建跨膜型Flt3L基因真核表達載體作為分子在佐劑,免疫印跡分析顯示體外轉(zhuǎn)染證實跨膜型Flt3L能在HeLa細胞中進行表達。然后,在C57BL/6小鼠中評價跨膜型Flt3L的佐劑活性。結(jié)果顯示,與單獨KAV肽相比較,將Flt3L分子佐劑與KAV聯(lián)合免疫,KAV肽疫苗特異性CD8~+T細胞的頻率明顯提高(P＜0.05),特異性CD8~+T細胞的表型有一定影響。 結(jié)論: 在成功克隆小鼠Flt3L基因的cDNA基礎(chǔ)上,構(gòu)建了小鼠Flt3L胞外域原核表達載體、可溶性Flt3L以及跨模型Flt3L真核表達載體,并證實能分別在大腸桿菌和HeLa細胞中表達。HeLa細胞中表達的可溶型Flt3L具有一定的生物學活性�？缒ば虵lt3L分子佐劑能夠顯著提高LCMV抗原肽特異性CD8~+ T細胞的頻率,并對特異性CD8~+T細胞的亞群有一定影響,提示該分子佐劑能促進肽疫苗的細胞免疫應(yīng)答。
[Abstract]:Objective: tumor and chronic virus infection is a major challenge to human beings, and immunotherapy is a promising new strategy for cancer and chronic virus infection. However, current clinical studies show that immunotherapy strategies, including therapeutic vaccines, have little effect. Therefore, finding new strategies to effectively enhance the efficacy of immunotherapy has become a hot topic. In this study, the effect of Ftl3L molecular adjuvant on the immune response of KAV peptide vaccine was studied using LCMV peptide vaccine as a model, and the mechanism of its action was preliminarily analyzed, which provided a reference for the clinical study of Flt3L molecular adjuvant. Methods: the existing Flt3L gene information was amplified by RT-PCR and cloned by RT-PCR. The prokaryotic expression plasmids of Flt3L, soluble and full-length Flt3L eukaryotic expression plasmids were constructed and verified by sequencing. The expression of E. coli or HeLa cells was detected by SDS-PAGE and / or Western blot. Then, after eukaryotic cell transfection, the biological activity of cell culture supernatant containing target protein was detected. The transmembrane Flt3L eukaryotic expression vector was used as a molecular adjuvant to evaluate its effect on the immune response induced by LCMV peptide vaccine in mice. Tetramer technique was used to analyze its effect on the frequency and phenotype of KAV-specific CD8T cells. Results: 1. The full-length cDNA of Flt3L coding region was obtained by RT-PCR and submitted to GenBank. the accession number was EU274583.Then, the extracellular domain prokaryotic expression vector of Flt3L was successfully constructed and sequenced correctly. It was confirmed that it was highly expressed in E. coli BL21 (DE3), and then the soluble Flt3L eukaryotic expression vector was constructed and sequenced, and then expressed in HeLa cells, and the supernatant of 48 h cell culture was obtained. The proliferation of mouse bone marrow cells and the colony culture of methyl cellulose semisolid culture were analyzed. The results showed that the culture supernatant transfected with soluble Flt3L had certain biological activity. It can promote colony formation induced by GM-CSF, and construct transmembrane eukaryotic expression vector of Flt3L gene as a molecular adjuvant. Western blot analysis showed that transmembrane Flt3L could be expressed in HeLa cells in vitro. Then, the adjuvant activity of transmembrane Flt 3 L was evaluated in C57BL / 6 mice. The results showed that the frequency of CD8T cells immunized with Flt3L molecular adjuvant combined with KAV peptide vaccine was significantly higher than that of KAV peptide alone (P < 0.05), and the phenotype of specific CD8T cells was affected to some extent. Conclusion: based on the successful cloning of mouse Flt3L gene cDNA, the mouse Flt3L extracellular prokaryotic expression vector, soluble Flt3L eukaryotic expression vector and transmodel Flt3L eukaryotic expression vector were constructed. It was also confirmed that soluble Flt3L expressed in E. coli and HeLa cells had certain biological activity. The transmembrane Flt3L molecular adjuvant could significantly increase the frequency of LCMV antigen peptide specific CD8T cells and influence the specific CD8T cell subsets, suggesting that this molecular adjuvant can promote the cellular immune response of peptide vaccine.
【學位授予單位】：暨南大學
【學位級別】：碩士
【學位授予年份】：2009
【分類號】：R392

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