副结核分枝杆菌MAP3061c蛋白表面展示疫苗载体的构建及其免疫效力研究

发布时间：2018-11-08 11:20

【摘要】：副结核分枝杆菌(Mycobacterium avium subsp.Paratuberculosis,MAP)是引发副结核病或者约内氏病(Johne’s disease,JD)的病原体,它主要感染反刍动物,造成慢性且无法治愈的肉芽肿肠炎,它的感染范围较广,包括家养的牛、羊及野生动物鹿等。由于患病动物的产奶能力和繁殖能力下降,对我国养殖业以及乳制品产业带来巨大的影响,造成巨大的经济损失,因此副结核病的防治已经成为我国养殖业所面临的重大问题。因而需要开发一种高效,经济,稳定的疫苗对副结核病进行控制。而表面展示疫苗在设计方面具有独特的优势,它将抗原递呈在细菌表面,这样使呈现在细菌表面的多肽抗原更容易被免疫系统识别。并且细菌的外膜蛋白和分泌的毒素都具有较强免疫原性,可以作为所展示的外源蛋白的免疫佐剂。因此,在新型疫苗的研制方面具有广阔的应用前景。本研究以冰核蛋白(Ice Nucleation Protein)作为展示平台,在大肠杆菌中构建表面展示系统。将目的基因克隆于构建的pET-INP表面展示载体,转入大肠杆菌BL21(DE3)中,IPTG诱导表达。经过Western-blot鉴定,亚细胞组分分离,蛋白酶K处理,斑点印迹鉴定,免疫荧光等实验方法,确定展示蛋白成功表达,并且展示在大肠杆菌表面。用成功构建的MAP3061c蛋白表面展示疫苗载体免疫Balb/C小鼠,定期采取血清、分离脾脏淋巴细胞,分别检测抗体水平、细胞因子水平、CD4+和CD8+细胞数量,分析体液与细胞免疫水平。然后进行攻毒实验,通过检测细胞因子变化,小鼠体重变化,以及小鼠结肠、肝、脾等病理变化评价免疫保护效果。结果表明,与对照组相比,重组菌免疫组抗体水平有较大程度提升,在三免后,其抗体水平为空载体对照组的4.2倍,显示该表面展示疫苗载体能刺激机体产生针对所展示蛋白的高水平特异性抗体;展示疫苗组的CD4+和CD8+T细胞显著增多,而且IFN-γ、IL-4、IL-23/IL-17等细胞因子水平显著升高,说明重组菌INP-MAP3061c能够介导机体产生较强的细胞免疫反应。在攻毒后,表面展示疫苗免疫组小鼠在攻毒16周之前增重率为增长状态,在16-20周为缓慢下降状态,但依然为正值,说明小鼠体重一直处于增长状态,没有消瘦。肝、脾等组织病理结果表明,MAP3061c蛋白表面展示疫苗可以降低病理损伤程度,减缓病程。综上,本研究以副结核分枝杆菌强免疫原性蛋白MAP3061c为对象,借助细菌表面展示平台,将其展示于大肠杆菌表面,所构建的表面展示活疫苗载体能够在小鼠中激发强烈的体液与细胞免疫,对副结核分枝杆菌感染具有较好的抵抗效果,为新型副结核疫苗的研制奠定基石,为副结核病的防控提供新思路。
[Abstract]:Mycobacterium paratuberculosis (Mycobacterium avium subsp.Paratuberculosis,MAP) is the pathogen that causes paratuberous or Johne's disease,JD disease. It mainly infects ruminants and causes chronic and incurable granulomatous enteritis. Including domestic cattle, sheep and wild animal deer and so on. Because the ability of producing milk and breeding of diseased animals has declined, it has brought great influence to the breeding and dairy industry of our country, and caused huge economic losses. Therefore, the prevention and treatment of paratuberous tuberculosis has become a major problem in the breeding industry of our country. Therefore, there is a need to develop an efficient, economical and stable vaccine for the control of paratuberculosis. The surface display vaccine has unique advantages in design. It presents the antigen on the bacterial surface, which makes it easier for the immune system to recognize the polypeptide antigen present on the bacterial surface. The outer membrane protein and toxin secreted by bacteria have strong immunogenicity and can be used as the immunoadjuvant of foreign proteins. Therefore, it has a broad application prospect in the development of new vaccines. In this study, the ice nucleoprotein (Ice Nucleation Protein) was used as the display platform to construct a surface display system in Escherichia coli. The target gene was cloned into the constructed pET-INP surface display vector and transferred into Escherichia coli BL21 (DE3) for IPTG induced expression. After Western-blot identification, subcellular component separation, protease K treatment, dot blot identification, immunofluorescence and other experimental methods, it was confirmed that the display protein was successfully expressed and displayed on the surface of Escherichia coli. Balb/C mice were immunized with successfully constructed MAP3061c protein surface display vaccine vector. Serum was used regularly to isolate spleen lymphocytes, antibody level, cytokine level, number of CD4 and CD8 cells were detected, humoral and cellular immunity levels were analyzed. The immune protective effect was evaluated by detecting the changes of cytokines, body weight, colon, liver, spleen and other pathological changes of mice. The results showed that, compared with the control group, the antibody level of the recombinant bacteria immunized group was increased to a great extent, and the antibody level of the recombinant bacteria immunized group was 4.2 times as high as that of the empty carrier control group after three immunizations. It is shown that the surface display vaccine vector can stimulate the body to produce a high level specific antibody against the displayed protein. The levels of CD4 and CD8 T cells and cytokines such as IFN- 纬 and IL-4,IL-23/IL-17 were significantly increased in the vaccine group, indicating that the recombinant strain INP-MAP3061c could induce a strong cellular immune response. After inoculation, the weight gain rate of the vaccinated mice was increased 16 weeks before the attack, and decreased slowly at 16-20 weeks, but it was still positive, indicating that the weight of the mice had been growing and no weight loss. The pathological results of liver and spleen showed that the surface display vaccine of MAP3061c protein could reduce the degree of pathological injury and slow down the course of disease. In this study, the strong immunogenicity protein (MAP3061c) of Mycobacterium paratuberculosis was used to display it on the surface of Escherichia coli with the help of bacterial surface display platform. The constructed surface display live vaccine vector can stimulate strong humoral and cellular immunity in mice and has good resistance to Mycobacterium paratuberculosis infection. To provide a new idea for the prevention and control of accessory tuberculosis.
【学位授予单位】：黑龙江八一农垦大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：S852.4

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