分支桿菌新型表達系統(tǒng)的建立及其在基因重組卡介苗研究中的應(yīng)用
發(fā)布時間：2019-06-20 07:01
【摘要】： 結(jié)核病是備受世界關(guān)注的一種主要公眾傳染性疾病。目前,全世界約有1/3人口(18.6億)攜帶有結(jié)核分支桿菌(Mycobacterium tuberculosis,M.tb),每年約有800萬新增病例,200萬人死于結(jié)核病。而且,結(jié)核病的控制也因為多重耐藥性(multidrug-resistant,MDR)菌株和艾滋病的出現(xiàn)使得本就十分嚴重的結(jié)核病疫情變得更加復(fù)雜化。目前,預(yù)防結(jié)核病唯一有效的疫苗是卡介苗(bacillusCalmette-Guerin,BCG),一種活的減毒牛型分支桿菌(M.bovis)。盡管BCG仍在許多國家廣泛用于兒童的免疫接種,但其對于成人肺結(jié)核的的保護效率仍一直存在很大的爭議。臨床試驗結(jié)果顯示,卡介苗對肺結(jié)核的免疫保護力介于0～80%之間,差異性極大。因此,研究一種保護效力超過BCG的結(jié)核病新疫苗勢在必行。 由于良好的免疫刺激效果以及廣泛使用的安全性能,使得BCG可作為預(yù)防結(jié)核病以及其他傳染病的優(yōu)良細菌表達載體。利用大腸桿菌-分支桿菌穿梭載體,不同病原體來源的保護性候選抗原均可在BCG中克隆并表達,從而構(gòu)建了相應(yīng)更為高效的重組BCG(rBCG)疫苗。然而,由于BCG生長緩慢、構(gòu)建的重組質(zhì)粒表達水平偏低等因素的影響,其應(yīng)用受到了一定的限制。近年來,隨著分子生物學(xué)和基因工程技術(shù)的發(fā)展,構(gòu)建可表達外源免疫優(yōu)勢抗原的rBCG研究發(fā)展迅速,并展現(xiàn)出了良好的應(yīng)用前景。本文旨在建立一套分支桿菌的高效表達系統(tǒng),以期實現(xiàn)目的基因在分支桿菌中的高水平表達;同時將其應(yīng)用于rBCG的研究中,構(gòu)建并篩選過表達結(jié)核桿菌嵌合抗原的基因重組卡介苗,并在動物水平上分析小鼠所誘導(dǎo)產(chǎn)生的抗原特異性細胞和體液免疫應(yīng)答效果以評估其免疫原性。 以恥垢分支桿菌(M.smegmatis)乙酰胺酶編碼基因啟動子(pACE)為基礎(chǔ)成功構(gòu)建了分支桿菌可控表達載體pMF系列,在蛋白水平驗證了pACE啟動子的調(diào)控嚴謹性,并成功的實現(xiàn)了M.tb嵌合抗原在M.smegmatis中的高水平表達;進一步分析其表達形式,發(fā)現(xiàn)主要為可溶性蛋白,從而免除了E.coli異源表達系統(tǒng)中所常見的蛋白變性與復(fù)性問題的困擾;另外,6×His Tag標簽的引入可方便的利用Ni~(2+)-NTA親和層析實現(xiàn)重組抗原的一步純化;嘗試將重組誘導(dǎo)表達載體轉(zhuǎn)化BCG,盡管加入誘導(dǎo)物,卻并未實現(xiàn)嵌合抗原在rBCG中的高水平表達,提示以pACE為基礎(chǔ)構(gòu)建的表達質(zhì)粒不適合作為rBCG的表達載體。 以E.coli lacZ為報告基因,在穿梭表達載體pMV261基礎(chǔ)上進行改造,構(gòu)建了分支桿菌啟動子探針載體pMC210;將結(jié)核桿菌鐵攝入蛋白上游調(diào)控序列/啟動子區(qū)域(pfurA)進行定點突變,并將pfurA及其突變體以基因融合的方式克隆于lacZ基因上游,通過β-半乳糖苷酶活性測定分析其啟動子強度。結(jié)果顯示,在兩種分支桿菌中(M.smegmatis和BCG),pfurA起始密碼子GTG→ATG突變(pfurAa)僅能引起大約2倍β-半乳糖苷酶活性的升高,AT富集區(qū)序列6-bp的替換突變(pfurAm)可使轉(zhuǎn)錄活性升高4～6倍;如果是上述兩者的聯(lián)合突變(pfurAma),β-半乳糖苷酶活性則升高約10倍,比在分支桿菌中過表達蛋白時常用的強啟動子phsp60也要高1.7～2倍。而且,有趣的是,pfurAs在慢速生長的BCG中比在快速生長的M.smegmatis中表現(xiàn)出了更高的β-半乳糖苷酶活性。 將帶有不同pfurA-lacZ融合片段的rBCG::lacZ菌株感染鼠巨噬細胞RAW264.7單細胞層,發(fā)現(xiàn)所有rBCG::lacZ菌株在感染初期,β-半乳糖苷酶表達均迅速上調(diào),1d后達到峰值;并可在細胞內(nèi)持續(xù)表達,7 d后酶活性仍可維持在較之體外略高的水平。隨后的動物實驗結(jié)果顯示,接種了rBCG::lacZ的小鼠成功的誘導(dǎo)出了增強的Th1型免疫應(yīng)答反應(yīng),主要表現(xiàn)為高滴度IgG2a的產(chǎn)生以及高水平IFN-γ的分泌,且其所誘導(dǎo)產(chǎn)生分泌IFN-γ的T淋巴細胞數(shù)量和rBCG::lacZ所表達的β-gal抗原水平呈正相關(guān)性。提示pfurAs啟動子系列非常適合在rBCG中驅(qū)動異源基因的表達,且以其為基礎(chǔ)構(gòu)建的重組卡介苗可誘導(dǎo)機體產(chǎn)生抗原特異性Th1為主的免疫應(yīng)答反應(yīng)。 因而,pfurA及其突變體被用來構(gòu)建分支桿菌的差異表達載體pMFA系列。應(yīng)用pMFA載體系列,成功的實現(xiàn)了M.tb嵌合抗原在M.smegmatis及BCG中以不同水平的差異性表達;具有起始密碼子及AT富集區(qū)聯(lián)合突變的pfurAma導(dǎo)致了最高水平的基因表達,這與β-半乳糖苷酶活性測定結(jié)果相一致。接著,以帶有不同pfurA-ag856a2融合片段的rBCG856A2免疫小鼠,進一步在動物水平驗證了該rBCG856A2可以在小鼠體內(nèi)分別誘導(dǎo)產(chǎn)生Ag85A、ESAT-6抗原特異的細胞免疫應(yīng)答和體液免疫應(yīng)答,說明嵌合基因rBCG856A2具有良好的免疫原性。另外,為了配合嵌合基因rBCG856A2的免疫檢測工作,我們還分別在E.coli中表達與純化了重組蛋白Ag85A、ESAT-6以及嵌合抗原Ag856A2,并同時制備了Anti-Ag85A及Anti-ESAT-6的小鼠單克隆抗體。接下來我們的工作重點將是進行小鼠攻擊試驗,以評估嵌合基因重組卡介苗的免疫保護效果,為日后的臨床試驗打下基礎(chǔ)。
[Abstract]:Tuberculosis is a major public infectious disease in the world. At present, about 1/3 of the world's population (18.6 billion) carries Mycobacterium tuberculosis (M.tb), about 8 million new cases per year, and 2 million deaths in tuberculosis. Moreover, the control of tuberculosis has also become more complicated by the emergence of multiple drug-resistant (MDR) strains and AIDS. At present, the only effective vaccine for the prevention of tuberculosis is the bacillusCalmette-Guerin (BCG), a live attenuated Mycobacterium bovis (M. bovis). Although BCG is still widely used in many countries for the immunization of children, the efficiency of its protection for adult tuberculosis continues to be a great deal. The results of the clinical trial show that the immune protection of BCG is between 0 and 80%, and the difference is great. Therefore, it is imperative to study a new vaccine for tuberculosis with more efficacy than BCG. The BCG can be used as an excellent bacterial expression for the prevention of tuberculosis and other infectious diseases due to the good immunostimulating effect and the safety performance that is widely used. The vector can be cloned and expressed in BCG by using the E. coli-mycobacterium shuttle vector and the protective candidate antigen of different pathogen sources, thereby constructing a corresponding more efficient recombinant BCG (rBCG). However, due to the slow growth of BCG and the low expression level of the recombinant plasmid, the application of the vaccine is limited. In recent years, with the development of molecular biology and genetic engineering technology, the development of rBCG, which can express exogenous immunodominant antigen, has been developed rapidly and has shown good application The aim of this paper is to establish a high-efficiency expression system of a set of mycobacteria, in order to realize the high-level expression of the target gene in the mycobacteria, and to construct and screen the gene recombination card expressing the chimeric antigen of the tubercle bacillus in the research of rBCG. The immune response effects of the antigen-specific cells and the humoral immune response induced by the mice were analyzed at the animal level to assess their immunity The expression vector pMF series was successfully constructed on the basis of the gene promoter (pACE) of the M. medegats, and the control of the pACE promoter was verified by the protein level, and the M. tb chimeric antigen was successfully realized in M. In addition, the introduction of the tag of the 6-His-His Tag is convenient to use the Ni ~ (2 +)-NTA affinity chromatography to realize the recombinant antigen. One-step purification; an attempt to transform the recombinant-induced expression vector into BCG, despite the addition of the inducer, did not achieve a high level of expression of the chimeric antigen in rBCG, suggesting that the expression plasmid constructed based on the pACE was not suitable as rBCG The expression vector was transformed with E. coli lacZ as the reporter gene on the basis of the shuttle expression vector pMV261, and the promoter probe vector pMC210 was constructed. A) a fixed point mutation is carried out, and the pfurA and the mutant thereof are cloned in the upstream of the lacZ gene in a gene fusion manner, The results show that the GTG-ATG mutation (pfurAa) of the pflA starting codon can only result in an increase in the activity of about 2-fold of the yeast-galactosylate enzyme, and the substitution mutation of the AT-rich region sequence of 6-bp (pfurAm) can make the transcription possible. The activity is increased by 4 to 6 times, and if the combination of the two is a joint mutation (pfurAma), the enzyme activity of the yeast-galactooligosaccharide is increased by about 10 times, and the strong promoter phsp60, which is commonly used when the protein is overexpressed in the branched bacterium, also It's about 1.7 to 2 times higher. And, interestingly, pfurAs shows a higher level in the slow-growing BCG than in the fast-growing M. medegats. -Galactomase activity. rBCG: lacZ strain with different pfurA-lacZ fusion fragments was infected with the mouse macrophage RAW264.7 single cell layer, and all rBCG: lacZ strains were found to be in the initial stage of infection, and the expression of the yeast-galactosylate enzyme was high. Up-regulated, peaked at 1 d, and can be continuously expressed in the cell, and the enzyme activity after 7d can still be maintained. The subsequent animal experiments showed that the mice inoculated with rBCG: lacZ successfully induced an enhanced Th1-type immune response, which mainly represented the production of high-titer IgG2a and the secretion of high-level IFN-1, and it induced the production of T-type of IFN-1. The number of cells and the amount of rBCG:1-g expressed by lacZ It is suggested that the pfurAs promoter series is very suitable for the expression of the heterologous gene in rBCG, and the recombinant BCG based on it can induce the antigen-specific T in the body. h1. Thus, pfurA and its mutants are used to construct the branch rod The expression vector pMFA was expressed by the differential expression vector pMFA. The pMFA vector series was used to successfully realize the differential expression of the M. tb chimeric antigen in M. smecatis and BCG at different levels. The pfatura with the combination mutation of the initiation codon and the AT-rich region resulted in the highest level of gene expression. The results showed that the rBCG856A2 with different pfurA-ag856a2 fusion fragment was used to immunize the mice, and the rBCG856A2 was further tested at the animal level. The specific cellular immune response and humoral immune response of Ag85A and ESAT-6 antigen could be induced in the mice, indicating that the chimeric gene rBCG The recombinant protein Ag85A, ESAT-6 and the chimeric antigen Ag856A2 were also expressed and purified in E. coli, and Anti-Ag85A and Anti-Ag856A2 were also prepared. The mouse monoclonal antibody of ESAT-6. Next, we will focus on the mouse attack test to assess the immune protective effect of the chimeric gene recombinant BCG
【學(xué)位授予單位】：復(fù)旦大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2008
【分類號】：R392
本文編號：2503023
[Abstract]:Tuberculosis is a major public infectious disease in the world. At present, about 1/3 of the world's population (18.6 billion) carries Mycobacterium tuberculosis (M.tb), about 8 million new cases per year, and 2 million deaths in tuberculosis. Moreover, the control of tuberculosis has also become more complicated by the emergence of multiple drug-resistant (MDR) strains and AIDS. At present, the only effective vaccine for the prevention of tuberculosis is the bacillusCalmette-Guerin (BCG), a live attenuated Mycobacterium bovis (M. bovis). Although BCG is still widely used in many countries for the immunization of children, the efficiency of its protection for adult tuberculosis continues to be a great deal. The results of the clinical trial show that the immune protection of BCG is between 0 and 80%, and the difference is great. Therefore, it is imperative to study a new vaccine for tuberculosis with more efficacy than BCG. The BCG can be used as an excellent bacterial expression for the prevention of tuberculosis and other infectious diseases due to the good immunostimulating effect and the safety performance that is widely used. The vector can be cloned and expressed in BCG by using the E. coli-mycobacterium shuttle vector and the protective candidate antigen of different pathogen sources, thereby constructing a corresponding more efficient recombinant BCG (rBCG). However, due to the slow growth of BCG and the low expression level of the recombinant plasmid, the application of the vaccine is limited. In recent years, with the development of molecular biology and genetic engineering technology, the development of rBCG, which can express exogenous immunodominant antigen, has been developed rapidly and has shown good application The aim of this paper is to establish a high-efficiency expression system of a set of mycobacteria, in order to realize the high-level expression of the target gene in the mycobacteria, and to construct and screen the gene recombination card expressing the chimeric antigen of the tubercle bacillus in the research of rBCG. The immune response effects of the antigen-specific cells and the humoral immune response induced by the mice were analyzed at the animal level to assess their immunity The expression vector pMF series was successfully constructed on the basis of the gene promoter (pACE) of the M. medegats, and the control of the pACE promoter was verified by the protein level, and the M. tb chimeric antigen was successfully realized in M. In addition, the introduction of the tag of the 6-His-His Tag is convenient to use the Ni ~ (2 +)-NTA affinity chromatography to realize the recombinant antigen. One-step purification; an attempt to transform the recombinant-induced expression vector into BCG, despite the addition of the inducer, did not achieve a high level of expression of the chimeric antigen in rBCG, suggesting that the expression plasmid constructed based on the pACE was not suitable as rBCG The expression vector was transformed with E. coli lacZ as the reporter gene on the basis of the shuttle expression vector pMV261, and the promoter probe vector pMC210 was constructed. A) a fixed point mutation is carried out, and the pfurA and the mutant thereof are cloned in the upstream of the lacZ gene in a gene fusion manner, The results show that the GTG-ATG mutation (pfurAa) of the pflA starting codon can only result in an increase in the activity of about 2-fold of the yeast-galactosylate enzyme, and the substitution mutation of the AT-rich region sequence of 6-bp (pfurAm) can make the transcription possible. The activity is increased by 4 to 6 times, and if the combination of the two is a joint mutation (pfurAma), the enzyme activity of the yeast-galactooligosaccharide is increased by about 10 times, and the strong promoter phsp60, which is commonly used when the protein is overexpressed in the branched bacterium, also It's about 1.7 to 2 times higher. And, interestingly, pfurAs shows a higher level in the slow-growing BCG than in the fast-growing M. medegats. -Galactomase activity. rBCG: lacZ strain with different pfurA-lacZ fusion fragments was infected with the mouse macrophage RAW264.7 single cell layer, and all rBCG: lacZ strains were found to be in the initial stage of infection, and the expression of the yeast-galactosylate enzyme was high. Up-regulated, peaked at 1 d, and can be continuously expressed in the cell, and the enzyme activity after 7d can still be maintained. The subsequent animal experiments showed that the mice inoculated with rBCG: lacZ successfully induced an enhanced Th1-type immune response, which mainly represented the production of high-titer IgG2a and the secretion of high-level IFN-1, and it induced the production of T-type of IFN-1. The number of cells and the amount of rBCG:1-g expressed by lacZ It is suggested that the pfurAs promoter series is very suitable for the expression of the heterologous gene in rBCG, and the recombinant BCG based on it can induce the antigen-specific T in the body. h1. Thus, pfurA and its mutants are used to construct the branch rod The expression vector pMFA was expressed by the differential expression vector pMFA. The pMFA vector series was used to successfully realize the differential expression of the M. tb chimeric antigen in M. smecatis and BCG at different levels. The pfatura with the combination mutation of the initiation codon and the AT-rich region resulted in the highest level of gene expression. The results showed that the rBCG856A2 with different pfurA-ag856a2 fusion fragment was used to immunize the mice, and the rBCG856A2 was further tested at the animal level. The specific cellular immune response and humoral immune response of Ag85A and ESAT-6 antigen could be induced in the mice, indicating that the chimeric gene rBCG The recombinant protein Ag85A, ESAT-6 and the chimeric antigen Ag856A2 were also expressed and purified in E. coli, and Anti-Ag85A and Anti-Ag856A2 were also prepared. The mouse monoclonal antibody of ESAT-6. Next, we will focus on the mouse attack test to assess the immune protective effect of the chimeric gene recombinant BCG
【學(xué)位授予單位】：復(fù)旦大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2008
【分類號】：R392
【引證文獻】
相關(guān)碩士學(xué)位論文 前2條
1 孫青;結(jié)核分枝桿菌無毒株H37Ra啟動子突變基因的比較分析[D];蘇州大學(xué);2010年
2 尹文東;結(jié)核分枝桿菌重組Ag85A、Ag85B蛋白聯(lián)合母牛分枝桿菌免疫原性的研究[D];吉林大學(xué);2012年
，本文編號：2503023
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