牛分枝杆菌保护性抗原多基因融合重组活载体的免疫分子机制研究

Mycobacterium bovis is the causive agent of bovine tuberculosis in a range of animal species and human beings,up to now, effective vaccination is not available. Although DNA vaccine has provided a new pathway for detection and prevention of bovine tuberculosis, there are many problems not solved in aspect of antigenic selection,immune pathway,immune programme and adjuvant. Recombinated adenovirus and lactobacillus have powerful dominance in activation of cell immunity and mucosae immunity. So in this study,based on the constructed eucaryotic expression recombinant of polygenic fusion and cytokine gene fusion, protective antigen fusion genes and cytokine fusion genes will be cloned into shuttle plasmid of adenovirus and expression vector of lactobacillus respectively, and then recombination adenovirus and lactobacillus of polygenic fusion for Mycobacterium bovis would be constructed succesfully. Through different immune pathway,immune schedule and conteracting toxic substances, we can analyze the immunologic mechanism and protection of recombinated adenovirus, recombinated lactobacillus and eukaryotic expression recombinants of polygenic fusion and cytokine fusion, furthermore, we can look for effective assortment of antigens and cytokines and lay solid foundation for developing new vaccine.

牛结核病主要是由牛分枝杆菌引起的人兽共患传染病，迄今尚无有效的预防疫苗。虽然DNA疫苗为牛结核病的检测和预防提供了新途径，但是，在抗原基因的选择、免疫途径、免疫程序、佐剂的选择等方面存在着尚待解决的问题。重组腺病毒、重组乳酸菌在激活机体细胞免疫和粘膜免疫方面有着强大的优势。因此，本研究在已构建的牛分枝杆菌保护性抗原多基因融合及与细胞因子基因融合真核表达重组质粒的基础上，获得牛分枝杆菌多保护性抗原融合基因及与细胞因子融合基因，克隆至腺病毒穿梭质粒及乳酸菌表达载体中，构建牛分枝杆菌保护性抗原多基因融合重组腺病毒和重组乳酸菌。通过不同的免疫途径、免疫方案，以及攻毒试验，研究重组腺病毒、重组乳酸菌，以及多价/与细胞因子融合真核表达重组质粒-重组活载体对实验动物的免疫分子机制和免疫保护作用，以期寻找有效抗牛分枝杆菌感染的保护性抗原基因组合或与细胞因子基因组合，为研发牛结核病新型疫苗奠定基础。

为开发牛结核病新型疫苗，探索腺病毒和乳酸菌作为牛分枝杆菌抗原表达和传递的载体。扩增了23个牛分枝杆菌保护性抗原基因及其融合基因，连接至腺病毒穿梭载体pacAd5CMVK-NpA和乳酸菌表达载体pNZ8149中，构建了13个重组穿梭质粒和11个重组表达质粒。重组穿梭质粒和pacAd5 9.2-100在AD293中包装了10株重组腺病毒。重组表达质粒转至NZ3900中获11株重组乳酸菌。以重组腺病毒、重组乳酸菌分别免疫小鼠，结果显示，与空载体对照组比较，重组腺病毒rAd5CMV-51、rAd5CMV-85a、rAd5CMV-51-63、rAd5CMV-83-85b、rAd5CMV-85a-70-γ组CD4+、CD8+T细胞明显增高。rAd5CMV-51、rAd5CMV-83、rAd5CMV-85a、rAd5CMV-85b、rAd5CMV-51-63、rAd5CMV-83-85b、rAd5CMV-85a-70-γ组分泌IFN-γ的细胞显著增高；rAd5CMV-85b、rAd5CMV-51-63、rAd5CMV-83-85b组分泌IL-2的细胞显著增高。rAd5CMV-83、rAd5CMV-85a组分泌IL-12的细胞显著增高。rAd5CMV-85a、rAd5CMV-85b、rAd5CMV-51-63、rAd5CMV-83-85b、rAd5 CMV-85a-70-γ、rAd5CMV-cfp10-esat6-cfp7组血清中特异性IgG水平显著增高。重乳酸菌pNZ8149-83-70/NZ3900组CD4+T细胞增高，pNZ8149-85a/NZ3900、pNZ8149-83-70/NZ3900 组CD8+T细胞明显增高。pNZ8149-85a/NZ3900、pNZ8149-83-70/NZ3900组分泌IFN-γ的水平显著增高。pNZ8149-83-70/NZ3900、pNZ8149-cfp10-esat6-cfp7/NZ3900组分泌IL-2的水平增高。pNZ8149-85a/NZ3900、pNZ8149-83-70/NZ3900组粪便中sIgA水平明显增高。结果表明，构建的重组腺病毒和重组乳酸菌能刺激免疫小鼠产生较高水平的细胞免疫、特异性体液免疫或粘膜免疫。为进一步开展牛体免疫试验奠定了基础，同时也为研制牛结核病新型疫苗提供了科学依据。