【作者】 靳彦文；

【导师】 马清钧； 曹诚；

【作者基本信息】 中国人民解放军军事医学科学院 ， 遗传学， 2005， 博士

【摘要】 DNA 疫苗又称核酸疫苗,作为一种新型疫苗,DNA疫苗与传统疫苗相比有着明显的优势,如易于生产,稳定性强,成本低廉等,并可同时诱导体液免疫和细胞免疫,但DNA疫苗的免疫效果在不同动物个体中差异较大,有些疫苗在小个体的动物中免疫和保护效果较好,而在大个体动物中效果较差,尤其是在大型动物和人类中,影响了它的实际应用。如何增强DNA疫苗的免疫反应成为当前研究的热点和难点。 我们以HBV DNA疫苗作为模型抗原,选择结核分支杆菌的热休克蛋白65、金黄色葡萄球菌肠毒素A和人胸腺素α原基因作为HBV DNA疫苗的佐剂,进行DNA疫苗免疫佐剂的研究。 本研究克隆了HBV的preS2S基因、结核分支杆菌的热休克蛋白65、金黄色葡萄球菌肠毒素A和人胸腺素α原基因,构建了三种单独表达的佐剂质粒:pHSP65、pmSEA、pThyα;三种融合表达的佐剂-抗原质粒:pHSP65/HBVs2s、pmSEA/HBVs2s、pThyα/HBVs2s;以及单独的模型抗原质粒pHBVs2s。体外瞬时转染293细胞或P815细胞,除外pmSEA/HBVs2s,Western Blot均检测到目的蛋白的表达,ELISA实验证实了转染细胞表达的HBsAg与相应的抗HBsAg抗体能产生良好的免疫反应,说明表达的重组抗原具有抗原性。 DNA疫苗与其佐剂免疫小鼠。ELISA结果显示三种佐剂均可增强pHBVs2s免疫小鼠抗HBs的产生,与单独pHBVs2s免疫组相比,提高了抗HBs抗体滴度。激发抗HBs抗体滴度的高低顺序依次是pHBVs2s+pThy α、pHBVs2s+pmSEA、pHBVs2s+pHSP65、pHBVs2s。ELISPOT检测分泌IFN-γ的脾淋巴细胞数量高低顺序依次是pHBVs2s+pmSEA、pHBVs2s+pThyα、pHBVs2s+pHSP65、pHBVs2s。HBsAg特异性CTLs的杀伤活性高低顺序分泌IFN-γ的脾淋巴细胞数量高低相一致。两种融合基因组小鼠也激发了抗HBs,其抗HBs抗体滴度的高低顺序依次是pThyα/HBVs2s和pHSP65/HBVs2s,pmSEA/HBVs2s未能诱导小鼠产生抗HBs抗体。HBsAg IgG亚类的分析发现:pThyα、pmSEA和pHSP65明显提高IgG2a,促进Th0向Th1分化。更多还原

【Abstract】 Over the past decade, genetic (or DNA) vaccines have emerged as a viable alternative approach to traditional vaccine, such as live-attenuated, subunit, peptide or protein vaccines. Intramuscular injection of DNA vaccine induces strong, long-lasting immune response to the antigen encoded by the gene subcloned into the plasmid. Plasmid DNA immunization leads to the production of antibody, activation of both . major histocompatibility complexes (MHC) class I-restricted cytotoxic T lymphocyte (CTL) and MHC class Ⅱ-restricted CD4+ T cells secreting Th1-type cytokines. It has potential advantages compared to traditional protein vaccine due to the prolonged antigen expression in the cytoplasm and loading of peptides derived from endogenous antigen onto class I MHC molecule, which is recognized by CTL. As a consequence, DNA vaccination has potential applications in the prevention or treatment of infectious diseases and cancers. However, results from clinical trials suggest that the immunogenicity of these vaccines in humans may be limited. Therefore, there is an urgent need for the development of potent and safe adjuvants to increase the activity of this class of vaccine, we and others have been exploring the benefit of co-immunizing with various types of vaccine adjuvant.HBV is a DNA virus with a partially single-stranded 3.2 kb genome encoding several viral proteins, including the core and envelope proteins. Of these proteins, the secreted middle ’"S" (surface) envelope protein is known to be highly immunogenic and can elicit both humoral and cell-mediated immune responses. So we choose the "preS2S" gene as our model antigen, then select prothymosin a (human) or staphylococcus enterotoxin A or heat shock protein 65 (tuberculosis) as an adjuvant improves antibody responses to HBV S antigen.In this study, we have constructed HBV DNA vaccine (pHBVs2s), and three plasmid containing gene of prothymosin α , staphylococcus enterotoxin A or heat shock protein 65 as gene adjuvant. We have also constructed three plasmid containinggene-fusious of prothymosin a . staphylococcus enterotoxin A or heat shock protein 65 with PS2S as DNA vaccine.When P815 (mastocytoma cell) or 293 cells were transfected with Flag- HBVs2s. Flag-pThy a/HBVS2S. Flag-pHSP65/HBVs2s, Flag- pmSEA/HBVs2s, Flag- pThy a, Flag-pmSEA. Flag-HSP65. or pcDNA3/flag vector by using lipofectamine 2000. Transfecting 24-48 hours, the cells were collected and analyzed by western blot and ELISA. Western blot assay showed that all the constructs except Flag-PS2S/PmSEA were expressed in P815 cells. The ELISA results confirmed that the cells which transfected with Flag-pHBVs2s, Flag-pThya/HBVS2S, Flag-pHSP65/HBVs2s have expressed HBsAg with corrective antigen- epitope.Anti-HBs antibody response was followed in the sera of mice injected with mixture of 100 ug of pcDNA3 , 100 u.g of pHBVS2S, a mixture of 100 ug of pThy a and 100 ug of pHBVS2S, a mixture of 100 |ag of pHSP65 and 100 ug of pHBVS2S or a mixture of 100 ug of pmSEA and 100 ug of pHBVS2S. After booster injection, mice inoculated with pHBVs2s+ pThy a generated the highest responses; a 5-fold increase in antibody titers relative to the response to the pHBVS2S alone was observed. Similarly, mice inoculated with pHBVs2s+ pmSEA or pHBVS2S+ pHSP65 generated greater responses compared to those inoculated with PreS2S alone.The ELISPOT assay was employed to determine the number of cells secreting interferon-y (IFN-y) upon in vitro re-stimulation with antigenic peptides. mice immunized with DNA encoding pHBVS2S and pThy a , pHBVS2S and pHSP65 or pHBVS2S+pmSEA generated ~3-fold more IFN-y ELISPOTs than mice immunized with the pHBVS2S alone did. Interestingly, the adjuvant also improved the readability of the ELISPOTs: both the size and intensity of spots was increased for IFN-y T cell responses to the HBV DNA vaccine.Three kinds of gene adjuvant boast the CTL activity induced by pHBVS2S. mice co-immunized with pHBVS2S and pThy a, pHBVS2S and pHSP65 or pHBVS2S+pmSEA exhibited greater cellular immune responses to HBsAg compared to those immunized with pHBVS2S alone, as determined by CTL activity of the splenocytes. Therefore, co-expression of pThy a, pHSP65 or pmSEA increased the更多还原