【作者】 蔡大川；

【导师】 任红；

【作者基本信息】 重庆医科大学 ， 内科学， 2004， 博士

【摘要】 目的：借助酵母菌的表达和免疫特性，利用基因工程技术构建表达乙型肝炎表面抗原及表面抗原与截短的热休克蛋白70的重组酵母菌，然后以整体活菌形式免疫BALB/C小鼠，作为一种针对HBV的治疗性疫苗，探索一种可有效活化机体抗HBV细胞免疫的免疫策略。方法：以基因工程技术构建内含HBsAg的重组啤酒酵母和毕赤酵母以及 HSP70（1-370）-HBsAg的重组毕赤酵母,以完整活菌形式于BALB/C小鼠皮下进行4次免疫后,用ELISA法检测其对乙型肝炎表面抗原的体液免疫及小鼠脾细胞与HBsAg共孵育后细胞因子的分泌情况；以[3H]掺入法测定小鼠脾细胞对HBsAg特异性的细胞增殖反应。结果：在两种重组酵母组中都成功诱导了小鼠体内乙型肝炎表面抗体的产生，显示乙型肝炎表面抗原抗原性并未受到影响。在重组啤酒酵母组中，通过[3H]掺入法可见HBsAg特异性的细胞增殖反应；在重组毕赤酵母组中，细胞因子检测结果显示重组毕赤酵母活菌可以诱导小鼠脾细胞分泌更多的IL-2，但对IL-4、γ-IFN的分泌无影响。结论：整体重组酵母活菌具有疫苗特性,可在小鼠体内激发体液免疫及一定程度的细胞免疫反应。重组酵母活菌用于激发机体抗HBV<WP=9>细胞免疫是一个全新的思路，重组酵母的用量、注射方式等有待进一步研究。更多还原

【Abstract】 Objective: Based on the expressing and immunologic characteristic of yeast, a new therapeutic vaccine, whole recombinant yeast expressing HBsAg or fusion protein of HBsAg and HSP70 (1-370), was used to explore a new way to activate cell-mediated anti-HBV immunity. Methods: The recombinant plasmids, pYES2-S, was constructed by inserting the HBsAg gene into the vector pYES2 .The recombinant plasmids, pPIC9K/S and PIC9K/hsp (1-370)-S, were constructed in a similar way. Then those recombinants were electro-transfected into Saccharomyces cerevisiae, INVSc-1, or Pichia pastoris, GS115, respectively. BALB/C mice were immunized with whole recombinant live yeast subcutaneously for 4 times. The humoral immunity to HBsAg was <WP=13>detected through ELISA assay of anti-HBsAg in mice’s serum; The ELISA assay was also used to detect the secretion of cytokine after the splenocytes were co-incubated with HBsAg for 72 hours. HBsAg-specific T cells proliferation was assayed though [3H] thymidine incorporation. Results: Recombinant whole live yeast successfully activated the humoral immunity, which showed that the reconstruction process had kept the antigenicity of HBsAg intact. On one hand, [3H] thymidine incorporation showed that there were HBsAg-specific T cells existing in mice immunized with whole recombinant Saccharomyces cerevisiae, on the other hand, the immunization with whole recombinant Pichia pastoris could facilitate the secretion of mIL-2 of those mice’ splenocytes. Conclusion: The whole recombinant live yeast may be used as therapeutic vaccine, but further study is needed for optimal way of immunization.更多还原