本文關(guān)鍵詞： 疫苗 Ag85A Ag85B 結(jié)核分枝桿菌 重組卡介苗　出處：《華中科技大學(xué)》2009年碩士論文　論文類型：學(xué)位論文

【摘要】：背景 疫苗是結(jié)核病(Tuberculosis,TB)預(yù)防策略中重要的醫(yī)學(xué)干預(yù)手段,卡介苗(Bacillus Calmette-Guérin, BCG)是目前世界上唯一被廣泛使用的預(yù)防結(jié)核疫苗,鑒于BCG對成人結(jié)核病保護(hù)效力的不穩(wěn)定,發(fā)展更好的抗結(jié)核疫苗成為迫切的需要。 重組卡介苗(rBCG)是一種很有前景的抗結(jié)核候選疫苗研制策略。抗原Ag85A和Ag85B均具有較強(qiáng)的免疫原性,并且是較有希望的結(jié)核病疫苗靶抗原;我們在前期的工作中發(fā)現(xiàn),編碼抗原Ag85A和Ag85B的基因疫苗免疫小鼠,可以刺激產(chǎn)生不同的免疫保護(hù)效應(yīng)。為了深入分析重組卡介苗的長期保護(hù)性和細(xì)胞免疫(CMI)的關(guān)系,在本課題的研究中,我們進(jìn)一步建立了分別過表達(dá)結(jié)核分枝桿菌(Mycobacterium tuberculosis,MTB)免疫優(yōu)勢抗原Ag85A或Ag85B的兩種重組卡介苗( rBCG::85A和rBCG::85B)菌株,并在小鼠體內(nèi)比較研究了兩種重組疫苗的保護(hù)性,為闡明重組卡介苗長期保護(hù)性的細(xì)胞免疫機(jī)制奠定基礎(chǔ)。 目的 1.建立重組卡介苗( rBCG::85A和rBCG::85B)菌株; 2.觀察rBCG誘導(dǎo)的免疫反應(yīng); 3.探討rBCG對小鼠結(jié)核模型的保護(hù)性與免疫保護(hù)機(jī)制。 方法 1.構(gòu)建表達(dá)Ag85A或Ag85B蛋白的重組大腸桿菌-分枝桿菌穿梭質(zhì)粒pMV85A, pMV85B;重組質(zhì)粒以及pMV261電穿法構(gòu)建重組卡介苗;Western blotting方法分析蛋白在rBCG中的表達(dá); 2. rBCG::85A和rBCG::85B免疫C57BL/6小鼠,以ELISA,FACS,ELISPOT等方法觀察特異性體液免疫和細(xì)胞免疫的產(chǎn)生; 3. rBCG::85A和rBCG::85B免疫BALB/c和C57BL/6小鼠,對免疫后小鼠進(jìn)行H37Rv毒株攻擊實(shí)驗(yàn),不同時(shí)間點(diǎn)測定組織荷菌量、組織病理學(xué)改變等指標(biāo)評價(jià)疫苗的保護(hù)性。 結(jié)果 1.成功構(gòu)建rBCG::85A和rBCG::85B;通過Western blotting方法對其菌體裂解上清和培養(yǎng)上清中蛋白的表達(dá)進(jìn)行鑒定,發(fā)現(xiàn),Ag85A蛋白作為一種分泌蛋白,主要表達(dá)于rBCG::85A菌株培養(yǎng)上清中,Ag85B蛋白主要存在于rBCG::85B菌株的培養(yǎng)上清中; 2. rBCG免疫C57BL/6小鼠后,能夠誘導(dǎo)特異性體液和細(xì)胞免疫的產(chǎn)生。ELISA結(jié)果顯示免疫小鼠可以產(chǎn)生高滴度的特異性抗體;IFN-γELISPOT實(shí)驗(yàn)示免疫組小鼠第6,24周,特異性蛋白刺激rBCG::85B和rBCG::85A組脾細(xì)胞,均產(chǎn)生較高的抗原特異的IFN-γ分泌細(xì)胞;FACS示肺臟CD4+/CD8+T細(xì)胞比例在rBCG::85B組增加,在rBCG::85A實(shí)驗(yàn)組減少;脾臟正好相反。 3.組織荷菌量顯示攻擊后4周,相比rBCG::261組,rBCG::85A和rBCG::85B組均可以明顯降低小鼠肺臟和脾臟載菌量,攻擊后18周,相比rBCG::85A組,rBCG::85B組降低肺臟載菌量更加顯著;肺臟組織病理學(xué)檢查結(jié)果示攻擊后18周,rBCG均可以提供比BCG更強(qiáng)的保護(hù)性。 結(jié)論 1.分別過表達(dá)結(jié)核分枝桿菌免疫優(yōu)勢抗原Ag85A和Ag85B的菌株重組卡介苗rBCG::85A和rBCG::85B;與原始卡介苗相比,這兩種重組卡介苗均能夠誘導(dǎo)較長期的保護(hù)性,rBCG::85B能夠誘導(dǎo)更強(qiáng)的保護(hù)性。 2.我們的結(jié)果顯示,重組卡介苗誘導(dǎo)的長期保護(hù)性跟抗原特異性IFN-γ相關(guān),并且肺臟早期的CD4+T細(xì)胞的應(yīng)答有可能與其更持久的保護(hù)性相關(guān)。
[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.

【學(xué)位授予單位】：華中科技大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2009
【分類號】：R392

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本文編號：1509219