【作者】 刘宏利；

【导师】 吴玉章；

【作者基本信息】 第三军医大学 ， 免疫学， 2003， 硕士

【摘要】 细胞毒性T淋巴细胞(cytotoxic T lymphocytes，CTLs)在机体抵抗肿瘤的过程中发挥着关键性的作用，激发体内有效的抗肿瘤CTL反应是目前肿瘤免疫治疗的主要目标。在当前的治疗方案中，由于肽疫苗和DNA疫苗具备较高的安全性和实用性，因而成为肿瘤疫苗学领域中发展最为迅速的肿瘤免疫治疗方案之一。但是这两种疫苗形式仍然具有各自的缺点，较低的免疫原性限制了肽疫苗的临床应用，以抗原多肽或重组蛋白进行免疫，通常不能激起机体有效的CTL反应，甚至还会导致机体对该抗原的特异性耐受；而DNA疫苗虽然可有效激发体内的CTL反应，但是其免疫反应强度通常较低，效应发挥滞后，不能提供较为有效的免疫保护。 大量的研究表明，多肽抗原与基因形式的抗原其抗原呈递动力学存在明显差异，肽抗原可直接快速呈递，无需表达过程，但是由于多肽的不稳定性，其呈递时效短暂；基因形式的抗原可被长期表达，其呈递具备持续性，但是由于抗原基因存在表达的过程，其呈递存在滞后效应。同时有研究表明，抗原CTL表位的递呈动力学参数是肿瘤特异性CTL活化的关键性因素。肽抗原和基因抗原呈递动力学的差异提示这两种抗原在激发机体免疫过程中可能存在互补性。 本课题以本研究所前期“模拟病毒”的研究工作为基础，对多肽疫苗和DNA疫苗进行综合，以期实现这两种抗原形式在抗肿瘤免疫激发过程中的协同互补，进而设计出肽-DNA复合疫苗。多聚赖氨酸作为自组织非病毒基因转染载体，已经在基因治疗领域得到广泛应用。在正负电荷相互吸引的静电作用下，富含正电荷的多聚赖氨酸可与富含负电荷的DNA分子结合，进而可以将质粒由几百纳米的松散线性分子压缩成直径数十纳米的致密颗粒。本研究以肿瘤相关抗原P815A为模式抗原，合成含有P815A CTL表位和多聚赖氨酸DNA结合区的双功能线性肽，并以该多肽对编码P815A和GM-CSF细胞因子的真核双表达质粒进行包装，进而形成肽-DNA复合疫苗颗粒。 本研究在技术路线上，以标准Fmoc方案化学合成上述双功能阳离子线性肽，中压液相色谱纯化，高压液相色谱、质谱鉴定；RT-PCR扩增鼠GM-CSF全长cDNA，常规基因重组技术构建P815A、GM-CSF双表达真核质粒；DNA沉淀分析、结合多肽增量分析、DNA凝胶阻滞、DNase Ⅰ保护试验及电镜分析优化疫苗颗粒制备条件；细胞转染、Western Blotting鉴定肽-DNA复合疫苗介导P815A、GM-CSF基因表达的有第二军医人学硕士研究生论文效性;’C:释放试验评价该疫苗激发体内CTL反应有效性;预防性保护试验、治疗性保护试验评价该疫苗激发体内抗瘤免疫的有效性。 结果显示，本研究所设计的阳离子双功能线性抗原肤被成功合成纯化;编码PS 15，、、GM一CSF的双表达真核质粒被正确构建;DNA沉淀分析、结合多肤增量分沂、DNA凝胶阻滞、ONasel保护试验及电镜分析确定了NaCI浓度87.5:二M、电荷比等于2的肤一DNA疫苗制备最优条件，并显示在该制备条件下质粒DNA被阳离子肤充分包装，并保护其免于DNasel的破坏;细胞转染、Western Blotting证实肤一DNA复合疫苗介导PS 15、、、GM一cSF基因表达的有效性;5‘Cr释放试验证实该疫苗激发体内P8!SA特异性CTL反应的有效性;预防性保护试验、治疗性保护试验显示该疫苗可预防性保护小鼠有效抵抗致命吐肿瘤，并可有效根治荷瘤小鼠的致死性Psls肿瘤。综上结果，本研究证实了!J太一 ONA复合疫苗被成功构建，初步证实了该疫苗激发抗瘤免疫的有效性，提示该肤一DNA复合疫苗有希望成为一种有效的肿瘤疫苗形式。本研究同时提示了!1太一从因抗原并供‘对于月中瘤疫苗设计的必要性和合理性。更多还原

【Abstract】 The cytotoxic T lymphocytes (CTLs) play a crucial role in anti-tumor immunity. The elicitation of strong CTL responses capable of mediating tumor regression in vivo has become the main aim of the immuotherpies for tumor. Peptide-based vaccines and naked DNA have been considered of the most rapidly evolving technologies for tumor vaccination due to their safety and feasibility. The major drawback Peptide-based vaccine is their weak inherent immunogenicity. Immunazition with peptide or recombinant proteins generally fails to elicit CTLs, and even in some case the induction of tolerance was observed. Although naked DNA vaccines in some cases may be effective in inducing immunity, however, the immune response that they elicit is often too weak or too late in developing to provide protective immunity.Previous studies demonstrated the differences between the dynamics for the presentations of these two forms of antigens. Antigenic peptides could be presented directly and quickly, however, for a short period of time due to their short half-life. Whereas, antigens in the form of genes could be expressed for a long time, but have a time lag owing to the process of gene expression. In addition, the dynamics of antigen presentation is thought a critical parameter in activating CTL response. Thus, the functional complementation of antigen forms of peptide and genes may be reasonably proposed and the collocation of antigenic peptide with gene encoding the same antigen might have a synergistic effect on elicitation of anti-tumor immunity.In the present study based on the previous research on "mimovirus" in our institute, we designed a peptide-DNA dual vaccine, which combines the peptide and DNA vaccines on the base of the probable functional complementation of antigen forms of peptide and genes. The cationic polylysine has been widely described as a self-assembly nonviral vector for gene therapy, which can condense plasmid DNA into small particles through electrostatic interactions between the positively charged lysine residues and the negatively charged DNA. As a test system, we chose a CTL epitope from the well-defined tumor Ag, P815A, as atarget and synthesized a bifunctional linear cationic peptide, which comprises a P815A CTL epitope and a DNA-binding moiety consisting of polylysine residues.In the present study, cationic antigenic peptide was synthesized by standard Fmoc, purified by middle-performance liquid chromatography and the purity was confirmed by reversed-phase high-performance liquid chromatography and the molecule weight by mass spectrometry. The gene for GM-CSF was amplified by RT-PCR and the plasmid encoding P815A and GM-CSF was constructed by standard recombinant technology. The condition of peptide-DNA vaccine preparation was determined by precipitation assay, gel retardation assay, DNase I protection assay and electronic microscopy. Further, the expressions of P815A and GM-CSF mediated by peptide-DNA vaccine particles were accessed by cell transfection and Western blotting. Finally, the efficacy of inducing antigen specific CTL response and eliciting anti-tumor immunity were accessed by 51Cr release assay and protection assay in P815 model.Here, we report that the cationic peptide was successfully synthesized and purified and the bicistronic plasmid encoding GM-CSF and P815A was constructed correctly. As bias of the results from precipitation, gel retardation, DNase I protection and electron microscopy assays, all vaccines used for immunization of animals were prepared by titrating cationic peptide into a solution of DNA with charge ratio of 2 at 87.5 mM NaCl concentration. The efficient expression of P815A and GM-CSF genes in cells mediated by peptide-DNA dual vaccine was conformed by cell transfection and Western blotting. The peptide-DNA dual vaccine elicited P815A specific CTL response, which was assayed in 51Cr release. Moreover, the peptide-DNA dual vaccine effectively protected DBA/2 mice from the fatal P815 tumor challenge and cure tumor-bearing DBA/2 mice with fatal P815 tumor,更多还原