Flt3L分子佐剂对LCMV肽疫苗特异性免疫应答的影响

发布时间：2018-06-17 20:35

  本文选题：Flt3L + 分子佐剂　； 参考：《暨南大学》2009年硕士论文

【摘要】： 目的: 肿瘤和慢性病毒感染是当前人类面临的重大挑战,而免疫治疗是针对肿瘤和慢性病毒感染的有前途的新策略。但是,现有的临床研究表明包括治疗性疫苗在内的免疫治疗策略的疗效甚微,因此,寻找如何有效增强免疫治疗效果的新策略已成为当前的研究热点。本研究以LCMV肽疫苗为模型,探讨Ftl3L分子佐剂对KAV肽疫苗的免疫应答的影响,并初步分析其作用机制,为Flt3L分子佐剂的临床研究提供参考。 方法: 通过对现有的Flt3L基因信息进行分析设计引物,RT-PCR法扩增并克隆其cDNA,进而分别构建Flt3L原核表达质粒、可溶型和全长Flt3L真核表达质粒,测序验证后,分别转化大肠杆菌或利用FuGENE HD试剂转染HeLa细胞进行表达,应用SDS-PAGE和/或免疫印迹检测其表达情况。然后,进行真核细胞转染后,对含有目标蛋白的细胞培养上清进行生物学活性检测。跨膜型Flt3L真核表达载体作为分子佐剂,在小鼠中评价其对LCMV肽疫苗诱发的免疫应答的影响,利用四聚体技术分析其对KAV特异性CD8~+T细胞频率与表型的干预作用。 结果: 1、通过RT-PCR获得与以往报道序列一致的Flt3L编码区全长cDNA,并已递交GenBank,登录号为EU274583;然后,成功构建了Flt3L的胞外域原核表达载体并测序正确,证实其能在大肠杆菌BL21(DE3)内高效表达。 2、进而构建可溶型Flt3L真核表达载体并测序验证正确,然后在HeLa细胞进行表达,获得48 h细胞培养上清。以小鼠骨髓细胞的增殖和甲基纤维素半固体培养集落培养实验进行分析,显示转染可溶型Flt3L的细胞培养上清具有一定的生物活性,能够促进GM-CSF引起的集落形成。 3、同时进一步构建跨膜型Flt3L基因真核表达载体作为分子在佐剂,免疫印迹分析显示体外转染证实跨膜型Flt3L能在HeLa细胞中进行表达。然后,在C57BL/6小鼠中评价跨膜型Flt3L的佐剂活性。结果显示,与单独KAV肽相比较,将Flt3L分子佐剂与KAV联合免疫,KAV肽疫苗特异性CD8~+T细胞的频率明显提高(P＜0.05),特异性CD8~+T细胞的表型有一定影响。 结论: 在成功克隆小鼠Flt3L基因的cDNA基础上,构建了小鼠Flt3L胞外域原核表达载体、可溶性Flt3L以及跨模型Flt3L真核表达载体,并证实能分别在大肠杆菌和HeLa细胞中表达。HeLa细胞中表达的可溶型Flt3L具有一定的生物学活性。跨膜型Flt3L分子佐剂能够显著提高LCMV抗原肽特异性CD8~+ T细胞的频率,并对特异性CD8~+T细胞的亚群有一定影响,提示该分子佐剂能促进肽疫苗的细胞免疫应答。
[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.
【学位授予单位】：暨南大学
【学位级别】：硕士
【学位授予年份】：2009
【分类号】：R392

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