比较研究过表达抗原Ag85A或Ag85B的重组卡介苗菌株的保护性

发布时间：2018-02-13 22:13

本文关键词： 疫苗 Ag85A Ag85B 结核分枝杆菌重组卡介苗　出处：《华中科技大学》2009年硕士论文　论文类型：学位论文

【摘要】：背景疫苗是结核病(Tuberculosis,TB)预防策略中重要的医学干预手段,卡介苗(Bacillus Calmette-Guérin, BCG)是目前世界上唯一被广泛使用的预防结核疫苗,鉴于BCG对成人结核病保护效力的不稳定,发展更好的抗结核疫苗成为迫切的需要。重组卡介苗(rBCG)是一种很有前景的抗结核候选疫苗研制策略。抗原Ag85A和Ag85B均具有较强的免疫原性,并且是较有希望的结核病疫苗靶抗原;我们在前期的工作中发现,编码抗原Ag85A和Ag85B的基因疫苗免疫小鼠,可以刺激产生不同的免疫保护效应。为了深入分析重组卡介苗的长期保护性和细胞免疫(CMI)的关系,在本课题的研究中,我们进一步建立了分别过表达结核分枝杆菌(Mycobacterium tuberculosis,MTB)免疫优势抗原Ag85A或Ag85B的两种重组卡介苗( rBCG::85A和rBCG::85B)菌株,并在小鼠体内比较研究了两种重组疫苗的保护性,为阐明重组卡介苗长期保护性的细胞免疫机制奠定基础。目的 1.建立重组卡介苗( rBCG::85A和rBCG::85B)菌株; 2.观察rBCG诱导的免疫反应; 3.探讨rBCG对小鼠结核模型的保护性与免疫保护机制。方法 1.构建表达Ag85A或Ag85B蛋白的重组大肠杆菌-分枝杆菌穿梭质粒pMV85A, pMV85B;重组质粒以及pMV261电穿法构建重组卡介苗;Western blotting方法分析蛋白在rBCG中的表达; 2. rBCG::85A和rBCG::85B免疫C57BL/6小鼠,以ELISA,FACS,ELISPOT等方法观察特异性体液免疫和细胞免疫的产生; 3. rBCG::85A和rBCG::85B免疫BALB/c和C57BL/6小鼠,对免疫后小鼠进行H37Rv毒株攻击实验,不同时间点测定组织荷菌量、组织病理学改变等指标评价疫苗的保护性。结果 1.成功构建rBCG::85A和rBCG::85B;通过Western blotting方法对其菌体裂解上清和培养上清中蛋白的表达进行鉴定,发现,Ag85A蛋白作为一种分泌蛋白,主要表达于rBCG::85A菌株培养上清中,Ag85B蛋白主要存在于rBCG::85B菌株的培养上清中; 2. rBCG免疫C57BL/6小鼠后,能够诱导特异性体液和细胞免疫的产生。ELISA结果显示免疫小鼠可以产生高滴度的特异性抗体;IFN-γELISPOT实验示免疫组小鼠第6,24周,特异性蛋白刺激rBCG::85B和rBCG::85A组脾细胞,均产生较高的抗原特异的IFN-γ分泌细胞;FACS示肺脏CD4+/CD8+T细胞比例在rBCG::85B组增加,在rBCG::85A实验组减少;脾脏正好相反。 3.组织荷菌量显示攻击后4周,相比rBCG::261组,rBCG::85A和rBCG::85B组均可以明显降低小鼠肺脏和脾脏载菌量,攻击后18周,相比rBCG::85A组,rBCG::85B组降低肺脏载菌量更加显著;肺脏组织病理学检查结果示攻击后18周,rBCG均可以提供比BCG更强的保护性。结论 1.分别过表达结核分枝杆菌免疫优势抗原Ag85A和Ag85B的菌株重组卡介苗rBCG::85A和rBCG::85B;与原始卡介苗相比,这两种重组卡介苗均能够诱导较长期的保护性,rBCG::85B能够诱导更强的保护性。 2.我们的结果显示,重组卡介苗诱导的长期保护性跟抗原特异性IFN-γ相关,并且肺脏早期的CD4+T细胞的应答有可能与其更持久的保护性相关。
[Abstract]:background
Is tuberculosis vaccine (Tuberculosis, TB) an important means of preventive medicine intervention strategy, BCG (Bacillus Calmette-Gu Rin, BCG) is the only thing in the world is widely used for prevention of tuberculosis vaccine, in view of the BCG effect on adult tuberculosis protection is not stable, anti tuberculosis vaccine development better has become an urgent need.
Recombinant BCG (rBCG) is a promising candidate for anti tuberculosis vaccine strategies. Antigens Ag85A and Ag85B have strong immunogenicity, and tuberculosis vaccine target antigen promising; we found in previous work, Ag85A and Ag85B antigen encoding gene vaccine in mice, can stimulate the immune protective effect of different. In order to long-term and in-depth analysis of cellular immune protection of recombinant BCG (CMI) relationship, in this study, we further established respectively the expression of Mycobacterium tuberculosis (Mycobacterium tuberculosis, MTB) two kinds of recombinant BCG immunodominant antigen Ag85A or Ag85B (85A and rBCG:: rBCG:: 85B) strains, and in vivo comparison of protective two recombinant vaccines, recombinant BCG to clarify the long-term protection of cellular immune system of the foundation.
objective
1. the recombinant Bacillus Calmette Guerin (rBCG:: 85A and rBCG:: 85B) was established.
2. the immune response induced by rBCG was observed.
3. to investigate the protective and immune protection mechanism of rBCG on mice tuberculosis model.
Method
1., we constructed a recombinant E.coli Mycobacterium shuttle plasmid pMV85A and pMV85B expressing Ag85A or Ag85B protein. Recombinant plasmid and pMV261 electroporation were used to construct recombinant BCG; Western blotting method was used to analyze the expression of protein in rBCG.
2. rBCG:: 85A and rBCG:: 85B immunized C57BL/6 mice with ELISA, FACS, ELISPOT and other methods to observe the production of specific humoral and cellular immunity.
3. rBCG:: 85A and rBCG:: 85B, immunizing BALB/c and C57BL/6 mice, carrying out H37Rv attack test on mice after immunization, measuring the amount of bacteria and histopathological changes at different time points, and evaluating the protection of vaccine.
Result
1. the successful construction of rBCG:: 85A and rBCG:: 85B by Western blotting method; cell lysates were analyzed and the expression of culture supernatant proteins were identified, found that the Ag85A protein as a secreted protein, mainly expressed in rBCG:: 85A strains in culture supernatant, Ag85B protein mainly exists in rBCG:: the culture supernatant of 85B strain in;
2. rBCG mice were immunized with C57BL/6, can produce.ELISA results in the induction of specific humoral and cellular immunity showed specific antibody immune mice can produce high titer; IFN- gamma ELISPOT assay indicated immunized mice at 6,24 weeks, specific protein stimulation of rBCG:: 85B and rBCG:: 85A group of spleen cells, IFN- had higher specific gamma the antigen producing cells; FACS showed the percentage of CD4+/CD8+T cells in lung rBCG:: 85B group, rBCG:: 85A in the experimental group decreased; spleen on the contrary.
The bacterial load of 3. group 4 weeks after the attack, compared to rBCG:: 261 groups, rBCG:: 85A and rBCG:: 85B group can significantly reduce the lung and spleen of mice carrying bacteria, 18 weeks after the attack of rBCG:: 85A group, rBCG: 85B group reduced lung microbial load is more significant; 18 weeks of lung tissue pathological examination results showed after the attack, rBCG can provide more protection than BCG.
conclusion
1., over expressing Mycobacterium tuberculosis immune predominance antigen Ag85A and Ag85B strain BCG rBCG:: 85A and rBCG:: 85B; compared with the original BCG, these two kinds of recombinant BCG can induce long-term protection, rBCG:: 85B can induce stronger protection.
2., our results showed that the long-term protection induced by recombinant BCG is associated with antigen specific IFN- gamma, and the response of CD4+T cells in the early stage of lung may be related to its more durable protection.

【学位授予单位】：华中科技大学
【学位级别】：硕士
【学位授予年份】：2009
【分类号】：R392

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