花生过敏原Arah2重组乳酸菌菌株的制备及其免疫保护机制的研究

Peanut allergy is the most common food allergy. Its incidence has been increasing in recent years and seriously affects the quality of life of some people and even life-threatening. There is no effective treatment to peanut allergy so far. Avoiding foods containing peanut allergen is the best choice for those who suffering peanut allergy. Therefore, it is necessary to explore new vaccines against peanut allergy. In this research, the mArah2 gene of peanut allergen was first synthesized artificially and then expressed in three different parts (intracellular, extracellular, and surface) of Lactococcus lactis, a kind of food-grade microorganism. The expression level of mArah2 protein was then compared among these recombinant strains. To assess the effects of these vaccines preventing peanut allergy, mice were first orally given the vaccines and then given peanut. The concentration of IgE, IgG, and IgA in serum and feces and cytokine levels in splenocyte supernatants were also measured by ELISA to evaluate the effects of these recombinant vaccines. To study and investigate the effects and mechanisms of these vaccines on peanut allergy, a murine model was established orally sensitized by peanut. The mice were then immunized with the three recombinant vaccines mentioned above. The behavior of mice were observed and recorded. The levels of IgE and IgG in serum and cytokine production in splenocyte supernatants were measured by ELISA. Flow cytometry was used to detect the numbers of CD4+CD25+Treg cells in spleen.

花生过敏是最常见的食物过敏，发病率逐年上升，严重影响部分人群的生活质量，甚至危及生命。目前尚无有效的治疗花生过敏的方法，避免摄入含有花生过敏原的食品是过敏患者的最佳选择。因此，研究一种有效的花生过敏疫苗，将为该病的防治提供思路和理论支持。本研究首先合成花生过敏原mArah2基因，将其分别表达于食品级微生物乳酸乳球菌的胞内、胞外和表面，构建重组花生过敏疫苗，并比较mArah2蛋白的表达量。为研究重组疫苗预防花生过敏的效果，给小鼠喂饲疫苗后再口服花生致敏，检测小鼠血液和粪便中IgE、IgG、IgA及脾细胞培养上清中细胞因子的含量。为考察重组疫苗是否能治疗花生过敏并探讨其发挥免疫作用的机制，建立小鼠花生致敏模型，将重组疫苗免疫过敏小鼠，观察小鼠的行为特征，ELISA法检测血清IgE、IgG和细胞因子的含量，流式细胞术测定脾脏CD4+CD25+Treg细胞的数量，评价所构建的疫苗的效果。

花生过敏是最常见的食物过敏，发病率逐年增加，目前尚无有效的治疗方法。因此，简单高效的预防性花生过敏疫苗的研制就显得尤为重要。本研究首先将花生过敏原Arah2的基因进行优化，并在食品级乳酸菌NZ9000的不同部位表达。成功表达外源蛋白的疫苗饲喂小鼠，与模型组相比，灌胃重组疫苗的小鼠能在一定程度上逆转Th2型过敏性应答（下调血清特异性IgE、脾细胞中细胞因子IL-4的水平）向Th1型保护性应答（血清特异性IgG2a、脾细胞中细胞因子IL-12、IN-γ及IFN-γ/IL-4比例）方向转变，且分泌表达和表面表达方式的疫苗的调节效果更加显著，优于胞内表达方式疫苗。为消除天然过敏原IgE表位带来的副反应，优化的过敏原基因进一步进行修饰，保留免疫原性而降低过敏原性。动物实验表明，该重组疫苗对花生过敏的预防作用更显著。.尘螨过敏疫苗的构建也基于同样的思路进行。与花生过敏疫苗不同的是，尘螨过敏原Derp2在乳酸菌中的表达部位对最终的小鼠体内免疫效果并无影响。由于本研究中所用的宿主菌乳酸乳球菌并无抗过敏特性，我们通过细胞实验筛查具有抗过敏特性的益生菌，并将其与重组疫苗混合，以研究混合物的预防效果。结果与重组疫苗并无显著差别，可能与目前较低的炎症水平有关。.发表SCI论文6篇，均为第一标注；中文CSCD收录论文2篇，分别为第一和第二标注；中文核心论文1篇，第一标注。申请与本项目相关的国家发明专利8项。

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