新型HIV多表位重组疫苗的构建、纯化及其实验免疫研究

发布时间：2018-07-07 15:47

本文选题：HIV + 表位　；参考：《吉林大学》2009年博士论文

【摘要】： 为研制安全有效的艾滋病治疗性疫苗,本研究在本室筛选出的一个较为成熟的多表位重组核酸疫苗(pVMEGNp24)的基础上引入了4-1BBL及OX40L两个协同刺激分子作为佐剂,构建了pVMEGNp24-4-1BBL-OX40L以及pVMEGNp24-OX40L。在哺乳动物细胞中表达的间接免疫荧光结果表明,所有构建的重组质粒编码蛋白均得到了正确表达。将己构建好的DNA疫苗(pVMEGNp24-41BBL-OX40L),经发酵罐建立发酵工艺,通过分子筛层析(Sepharose 6 FF)去除RNA,亲和层析(PlasmidSelect Xtra)去除线性质粒DNA,阴离子交换层析(SOURCE30Q)精纯,以中空纤维交叉流系统浓缩至药用剂量。经过质量鉴定结果表明,该纯化质粒疫苗临床用质粒DNA的质量标准,所建立的工艺可行。将纯化后的重组DNA(rDNA)疫苗质粒通过导入人外周血来源的DC。并将转染外源基因的成熟DC细胞与自体淋巴细胞共培养。免疫学指标检测结果表明:协同刺激分子在诱导细胞免疫应答上作用强大,并且协同刺激分子的联合应用具有明显的协同累积作用。采用rDNA/rFPV(重组鸡痘病毒)联合免疫策略,免疫中国恒河猴,以人-猴艾滋病重组病毒SHIV-KB9攻毒后,分析疫苗免疫原性和攻毒保护效果。结果表明:协同刺激分子可以诱导免疫动物产生更高水平的细胞及体液免疫反应和较强的记忆性免疫反应。SHIV-KB9病毒攻击后,单协同刺激分子免疫组的动物获得的保护效果更好,单协同刺激分子疫苗是一个有潜力的候选疫苗。为HIV重组疫苗的研制与临床前实验研究奠定了基础。
[Abstract]:In order to develop a safe and effective AIDS therapeutic vaccine, a mature multiepitope recombinant nucleic acid vaccine (pVMEGNp24) was selected in this study. Two co-stimulant molecules, 4-1BBL and OX40L, were introduced as adjuvants to construct pVMEGNp24-4-1BBL-OX40L and pVMEGNp24-OX40L. The results of indirect immunofluorescence in mammalian cells showed that all the constructed recombinant plasmid encoded proteins were correctly expressed. The constructed DNA vaccine (pVMEGNp24-41BBL-OX40L) was fermented in fermenter. The RNAs were removed by Sepharose 6FF, the linear plasmid DNA was removed by PlasmidSelect Xtra, the anion exchange chromatography (SOURCE30Q) was purified and concentrated to medical dosage by hollow fiber cross flow system. The results of quality identification showed that the quality standard of plasmid DNA for clinical use of the purified plasmid vaccine was feasible. The purified recombinant DNA (rDNA) vaccine plasmid was transfected into DC1 from human peripheral blood. Mature DC cells transfected with exogenous genes were co-cultured with autologous lymphocytes. The results of immunological indices showed that the co-stimulator had a strong effect on inducing cellular immune response, and the combined application of co-stimulator had obvious synergistic cumulative effect. RDNA / rFPV (recombinant fowlpox virus) was used to immunize Chinese rhesus monkeys. The immunogenicity and protective effect of rDNA rFPV (recombinant fowlpox virus) vaccine against human monkey AIDS recombinant virus SHIV-KB9 were analyzed. The results showed that the co-stimulator could induce higher level of cellular and humoral immune response and stronger memory immune response of immunized animals, and the protective effect of the single co-stimulatory molecular immunization group was better than that of the control group. Single costimulatory molecular vaccine is a potential candidate vaccine. It lays a foundation for the development of recombinant HIV vaccine and preclinical experimental research.
【学位授予单位】：吉林大学
【学位级别】：博士
【学位授予年份】：2009
【分类号】：R392

【引证文献】