鼠疫耶尔森氏菌新型候选DNA疫苗的构建及其免疫效果鉴定

发布时间：2018-04-28 15:32

本文选题：鼠疫耶尔森氏菌 + F1-V　；参考：《第三军医大学》2005年硕士论文

【摘要】：鼠疫杆菌(Yersinia pestis)是腺鼠疫、败血症鼠疫和肺鼠疫的自然疫源性病原,曾经以“瘟疫”的形式给人类的健康造成了极大的危害,历史上共造成约2 亿人死亡,现在世界各地每年仍有鼠疫病例报道。由于生物恐怖袭击事件的发生,已引起世界各国对鼠疫的高度警惕。恐怖袭击常采用气溶胶感染,可直接引起原发性肺鼠疫流行,因而更需要研究安全有效的鼠疫疫苗。传统的鼠疫疫苗包括灭活全菌体死疫苗、减毒活疫苗。虽然灭活全菌体死疫苗在预防腺型鼠疫有一定效果,但对肺型鼠疫无效;减毒活疫苗又具有潜在的毒性。近年来,DNA 疫苗由于能够诱导出较好的体液免疫和细胞免疫而日益受到重视,被誉为疫苗的第三次革命。tPA 信号肽可以促进异种蛋白的分泌表达,增强表达抗原的免疫原性。本文对鼠疫耶尔森氏菌新型DNA 疫苗进行了初步研究,选取鼠疫菌保护性抗原F1、V 蛋白编码基因和人tPA 信号肽为研究对象,PCR 扩增鼠疫菌F1 和V 编码基因,分别与克隆载体pGEM-T 连接后测序,将测序正确的片段与真核表达质粒pVAX1连接,分别构建重组真核表达质粒pVAX1/F1、pVAX1/V 和pVAX1/F1-V,扩增tPA 信号肽,分别插入到F1、V 和F1-V 目的基因的上游,构建分泌型重组真核表达质粒tPA-pVAX1/F1、tPA-pVAX1/V 和tPA-pVAX1/F1-V。重组质粒转染COS-7 细胞,细胞免疫化学方法和Western blot 方法鉴定结果显示重组质粒可表达F1、V 和F1-V 蛋白。在此基础上,重组质粒分别加细胞因子佐剂GM-CSF 免疫BALB/c 小鼠,每组10只,并设PBS 组为阴性对照,100μg/只,双侧股四头肌肌肉注射,隔周免疫一次,加强免疫3 次,检测细胞免疫和体液免疫指标,ELISA 方法检测小鼠血清中总IgG 水平,免疫组显著高于PBS 对照组(P0.01),在初次免疫后tPA-pVAX1/V 和tPA-pVAX1/F1-V组可以诱导出较高的抗体水平,而其它组要至少二次免疫后才能达到相应水平。抗体亚型分类检测结果显示tPA-pVAX1/V 和tPA-pVAX1/F1-V 组的IgG2a 水平显著提高,说明分泌型tPA-V 和tPA-F1-V DNA疫苗免疫在机体内成功地增强了Th1型细胞免疫为主的免疫应答。ELISPOT 方法检测了特异性活化的可以分泌IFN-γ的CD8+ T 淋巴细胞数,免疫组小鼠形成斑点的细胞数均显著多于PBS 对照组(P0.01),以tPA-pVAX1/V 和
[Abstract]:Yersinia pestis (Yersinia pestis) is the natural pathogen of plague, septicemia and pneumonic plague, which has caused great harm to human health in the form of "plague", and has caused a total of 200 million deaths in history. Plague cases are still reported every year around the world. Due to the occurrence of bioterrorist attacks, countries around the world have been highly alert to plague. Aerosol infection is often used in terrorist attacks, which can directly cause the epidemic of primary pneumonic plague, so it is necessary to study a safe and effective plague vaccine. Traditional Yersinia pestis vaccine includes inactivated whole cell dead vaccine and attenuated live vaccine. Although the inactivated whole cell death vaccine has certain effect in preventing glandular plague, it has no effect on pulmonary plague, and attenuated live vaccine has potential toxicity. In recent years, DNA vaccine has been paid more and more attention because of its ability to induce better humoral and cellular immunity. It is known as the third revolution of vaccine. TPA signal peptide can promote the secretion and expression of xenogeneic proteins and enhance the immunogenicity of expressed antigens. A novel Yersinia pestis DNA vaccine was studied in this paper. The gene encoding F1V protein of Yersinia pestis protection antigen and human tPA signal peptide were selected to amplify the F1 and V coding genes of Yersinia pestis. The recombinant eukaryotic expression plasmids pVAX1 / F1pVAX1 / V and pVAX1 / F1-V were constructed, and the tPA signal peptides were amplified and inserted into the upstream of F1V and F1-V target genes, respectively. The secretory recombinant eukaryotic expression plasmids tPA-pVAX1 / F1 + tPA-pVAX1 / V and tPA-pVAX1 / F1-V were constructed. The recombinant plasmid was transfected into COS-7 cells. The results of cell immunocytochemistry and Western blot analysis showed that the recombinant plasmid could express F1V and F1-V proteins. On this basis, 10 BALB/c mice were immunized with recombinant plasmids with cytokine adjuvant GM-CSF, 10 mice in each group were immunized with PBS group as negative control (100 渭 g / mouse), bilateral quadriceps femoris were injected intramuscularly, once every other week, and 3 times were strengthened. The levels of total IgG in serum of mice were detected by Elisa. The levels of total IgG in the immunized group were significantly higher than those in the PBS control group (P 0.01). The higher antibody level could be induced in the tPA-pVAX1/V and tPA-pVAX1/F1-V groups after the first immunization. Other groups had to be immunized at least twice before they reached the level. The results of classification of antibody subtypes showed that the level of IgG2a in tPA-pVAX1/V and tPA-pVAX1/F1-V groups was significantly increased. The results showed that the secretory tPA-V and tPA-F1-V DNA vaccine immunization successfully enhanced the immune response of Th1 type cellular immunity. Elispot method detected the number of specifically activated CD8 T lymphocytes which could secrete IFN- 纬. The number of cells forming spots in immunized mice was significantly higher than that in PBS control group (P 0.01).
【学位授予单位】：第三军医大学
【学位级别】：硕士
【学位授予年份】：2005
【分类号】：R392

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