【作者】 赵丽娟；

【导师】 房明丽；

【作者基本信息】 吉林大学 ， 生物化学与分子生物学， 2022， 硕士

【摘要】 鲍曼不动杆菌对临床大多数抗生素存在耐药性,已成为最常见的“超级细菌”之一,常引起呼吸道感染、菌血症、脑膜炎等严重疾病。因此,预防鲍曼不动杆菌的传播与感染迫在眉睫,开发疫苗则是最有效的手段。目前已有针对鲍曼不动杆菌的灭活疫苗、DNA疫苗和重组亚单位疫苗等正在研究中,但未有一种疫苗进入临床试验阶段。而且,现有的疫苗存在着周期长、免疫原性弱、安全性低等缺点,急需开发有效的新型疫苗用于防治鲍曼不动杆菌的感染。本课题组前期工作开发了重组减毒沙门氏菌疫苗载体χ9241,其可携带外源抗原定植到机体免疫器官,且自带佐剂特性,能诱导强大的免疫反应。因此,本研究针对鲍曼不动杆菌开发了一种基于χ9241的细菌载体疫苗用于预防该细菌感染。本研究根据鲍曼不动杆菌的生物学特点及外膜结构,选择了以外膜蛋白A(OmpA)作为其疫苗抗原靶点,并根据OmpA蛋白的抗原表位分析结果,分别设计了蛋白疫苗Ab TompA和减毒沙门氏菌载体疫苗χ9241-rompA。为了达到最佳的免疫效果,本研究选择了序贯加强免疫策略,即χ9241-rompA与Ab TompA疫苗的联合免疫策略,结合这两种疫苗的优势,以期望诱导强大的免疫反应来保护小鼠抵抗鲍曼不动杆菌的攻击。本研究的主要实验内容及结果如下:1.采用生物信息学方法分析鲍曼不动杆菌OmpA的表位,分别构建了鲍曼不动杆菌全长OmpA蛋白(Ab FompA)、鲍曼不动杆菌OmpA蛋白疫苗(Ab TompA)及携带重组OmpA质粒的减毒沙门氏菌载体疫苗(χ9241-rompA)。其中,Ab FompA用于免疫家兔来获得阳性血清用于后续的蛋白鉴定。2.采用序贯加强免疫策略,将χ9241-rompA与Ab TompA联合免疫BALB/c小鼠,评价该策略在小鼠体内诱导的免疫反应。结果发现,联合疫苗组与单一疫苗组χ9241-rompA或Ab TompA相比,可诱导更高水平的抗OmpA总Ig G和粘膜IgA,并诱导更平衡的Th1/Th2免疫反应,同时高效刺激脾细胞的增殖,产生更高水平的IFN-γ。所有疫苗组均能活化DC、B细胞,提高CD4+T细胞和Th17细胞的比例。3.与单一疫苗组相比,χ9241-rompA与Ab TompA联合免疫组血清能更显著的抑制鲍曼不动杆菌的生物活性,包括黏附、迁移和生物被膜的形成。4.本研究分别以鲍曼不动杆菌通过腹腔和鼻腔感染小鼠后引发的系统性脓毒症和肺部炎症为模型,在小鼠体内评估疫苗是否能抵抗细菌标准株和耐药株的攻击。结果发现,χ9241-rompA与Ab TompA联合免疫组可以提高小鼠生存期,降低血清细胞因子IL-6的水平,减轻肺部组织病理损伤。本研究不仅为鲍曼不动杆菌的疫苗研究提供了多种候选疫苗,也为沙门氏菌载体的开发及序贯加强免疫策略的应用提供了新的实验基础。更多还原

【Abstract】 Acinetobacter baumannii has become one of the most common "super bacteria" due to its resistance to most antibiotics,it often causes serious diseases such as respiratory tract infection,bacteremia,and meningitis.Therefore,there is urgent for taking solutions to prevent the transmission and infection ofAcinetobacter baumannii,while the development of vaccines is the most effective way.Currently,the inactivated vaccines,DNA vaccines and recombinant subunit vaccines againstAcinetobacter baumannii are under study,but none of them have entered the stage of clinical trials.Moreover,the existing vaccines have a lot of disadvantages such as the long cycle,weak immunogenicity and low safety,so,it need to develop effective vaccines for the prevention ofAcinetobacter baumannii infection.In the previous work of our research group,an attenuated Salmonella vector vaccine χ9241 was developed,which has adjuvant property,and can carry foreign antigens to colonize the immune organs of the body to induce a strong immune response.Therefore,in this study,we developed a vaccine based on χ9241 againstAcinetobacter baumannii infection.In this study,according to the biological characteristics and outer membrane structure ofAcinetobacter baumannii,the outer membrane proteinA(OmpA)was selected as the vaccine antigen,and a protein vaccineAb TompA and a recombinant attenuated Salmonella vector vaccine χ9241-rompA were designed according to the epitope analysis results of OmpA protein.In order to get the stronger immunoprotection,we choose a heterologous prime-boost strategy,combining the χ9241-rompA andAb TompA vaccine,expecting to induce a stronger immune response to protect mice against challenge withAcinetobacter baumannii.The main experimental contents and results of this study are as follows:1.The epitope ofAcinetobacter baumannii OmpA was analyzed by bioinformatics method,and the full-length OmpA protein ofAcinetobacter baumannii(Ab FompA),theAcinetobacter baumannii OmpA protein vaccine(Ab TompA)and the recombinant attenuated Salmonella vector vaccine(χ9241-rompA)were constructed respectively.Notably,Ab FompA was used to immunize rabbit to get positive serum for subsequent protein identification assays.2.χ9241-rompA andAb TompA were combined to immunize BALB/c mice to evaluate the immune response induced by prime-boost strategy in mice.Compared to the χ9241-rompA orAb TompA vaccine,the combined vaccines could induce higher levels of anti-OmpA total Ig G and mucosal IgA,and also induce a more balanced Th1/Th2 immunity.Moreover,the more higher cell proliferation ratio and the production of IFN-γ were observed in mouse splenocytes after theAb FompA stimulation.Meanwhile,all vaccine groups could activate DC,B cells,and increase the ratio of CD4+ T cells and Th17 cells.3.Compared to the χ9241-rompA orAbTompA vaccine,the serum of the combined vaccines could significantly inhibit the biological activities ofAcinetobacter baumannii,including the abilities of adhesion,migration and biofilm formation.4.Acinetobacter baumannii-induced systemic sepsis and pulmonary inflammation models were used to evaluate the effect of the vaccine protection against challenge with standard strains and drug-resistant strains in mice.The results showed that the combined immunization group of χ9241-rompA andAb TompA could improve the survival time of mice,reduce the level of serum cytokine IL-6,and alleviate the pathological damage of lung tissue.Collectly,this study not only provides a variety of candidate vaccines forAcinetobacter baumannii research,but also provides a new experimental basis for the development of recombinant attenuated Salmonella vector and the application of primeboost immunization strategy.更多还原