分支杆菌新型表达系统的建立及其在基因重组卡介苗研究中的应用
发布时间：2019-06-20 07:01
【摘要】： 结核病是备受世界关注的一种主要公众传染性疾病。目前,全世界约有1/3人口(18.6亿)携带有结核分支杆菌(Mycobacterium tuberculosis,M.tb),每年约有800万新增病例,200万人死于结核病。而且,结核病的控制也因为多重耐药性(multidrug-resistant,MDR)菌株和艾滋病的出现使得本就十分严重的结核病疫情变得更加复杂化。目前,预防结核病唯一有效的疫苗是卡介苗(bacillusCalmette-Guerin,BCG),一种活的减毒牛型分支杆菌(M.bovis)。尽管BCG仍在许多国家广泛用于儿童的免疫接种,但其对于成人肺结核的的保护效率仍一直存在很大的争议。临床试验结果显示,卡介苗对肺结核的免疫保护力介于0～80%之间,差异性极大。因此,研究一种保护效力超过BCG的结核病新疫苗势在必行。 由于良好的免疫刺激效果以及广泛使用的安全性能,使得BCG可作为预防结核病以及其他传染病的优良细菌表达载体。利用大肠杆菌-分支杆菌穿梭载体,不同病原体来源的保护性候选抗原均可在BCG中克隆并表达,从而构建了相应更为高效的重组BCG(rBCG)疫苗。然而,由于BCG生长缓慢、构建的重组质粒表达水平偏低等因素的影响,其应用受到了一定的限制。近年来,随着分子生物学和基因工程技术的发展,构建可表达外源免疫优势抗原的rBCG研究发展迅速,并展现出了良好的应用前景。本文旨在建立一套分支杆菌的高效表达系统,以期实现目的基因在分支杆菌中的高水平表达;同时将其应用于rBCG的研究中,构建并筛选过表达结核杆菌嵌合抗原的基因重组卡介苗,并在动物水平上分析小鼠所诱导产生的抗原特异性细胞和体液免疫应答效果以评估其免疫原性。 以耻垢分支杆菌(M.smegmatis)乙酰胺酶编码基因启动子(pACE)为基础成功构建了分支杆菌可控表达载体pMF系列,在蛋白水平验证了pACE启动子的调控严谨性,并成功的实现了M.tb嵌合抗原在M.smegmatis中的高水平表达;进一步分析其表达形式,发现主要为可溶性蛋白,从而免除了E.coli异源表达系统中所常见的蛋白变性与复性问题的困扰;另外,6×His Tag标签的引入可方便的利用Ni~(2+)-NTA亲和层析实现重组抗原的一步纯化;尝试将重组诱导表达载体转化BCG,尽管加入诱导物,却并未实现嵌合抗原在rBCG中的高水平表达,提示以pACE为基础构建的表达质粒不适合作为rBCG的表达载体。 以E.coli lacZ为报告基因,在穿梭表达载体pMV261基础上进行改造,构建了分支杆菌启动子探针载体pMC210;将结核杆菌铁摄入蛋白上游调控序列/启动子区域(pfurA)进行定点突变,并将pfurA及其突变体以基因融合的方式克隆于lacZ基因上游,通过β-半乳糖苷酶活性测定分析其启动子强度。结果显示,在两种分支杆菌中(M.smegmatis和BCG),pfurA起始密码子GTG→ATG突变(pfurAa)仅能引起大约2倍β-半乳糖苷酶活性的升高,AT富集区序列6-bp的替换突变(pfurAm)可使转录活性升高4～6倍;如果是上述两者的联合突变(pfurAma),β-半乳糖苷酶活性则升高约10倍,比在分支杆菌中过表达蛋白时常用的强启动子phsp60也要高1.7～2倍。而且,有趣的是,pfurAs在慢速生长的BCG中比在快速生长的M.smegmatis中表现出了更高的β-半乳糖苷酶活性。 将带有不同pfurA-lacZ融合片段的rBCG::lacZ菌株感染鼠巨噬细胞RAW264.7单细胞层,发现所有rBCG::lacZ菌株在感染初期,β-半乳糖苷酶表达均迅速上调,1d后达到峰值;并可在细胞内持续表达,7 d后酶活性仍可维持在较之体外略高的水平。随后的动物实验结果显示,接种了rBCG::lacZ的小鼠成功的诱导出了增强的Th1型免疫应答反应,主要表现为高滴度IgG2a的产生以及高水平IFN-γ的分泌,且其所诱导产生分泌IFN-γ的T淋巴细胞数量和rBCG::lacZ所表达的β-gal抗原水平呈正相关性。提示pfurAs启动子系列非常适合在rBCG中驱动异源基因的表达,且以其为基础构建的重组卡介苗可诱导机体产生抗原特异性Th1为主的免疫应答反应。 因而,pfurA及其突变体被用来构建分支杆菌的差异表达载体pMFA系列。应用pMFA载体系列,成功的实现了M.tb嵌合抗原在M.smegmatis及BCG中以不同水平的差异性表达;具有起始密码子及AT富集区联合突变的pfurAma导致了最高水平的基因表达,这与β-半乳糖苷酶活性测定结果相一致。接着,以带有不同pfurA-ag856a2融合片段的rBCG856A2免疫小鼠,进一步在动物水平验证了该rBCG856A2可以在小鼠体内分别诱导产生Ag85A、ESAT-6抗原特异的细胞免疫应答和体液免疫应答,说明嵌合基因rBCG856A2具有良好的免疫原性。另外,为了配合嵌合基因rBCG856A2的免疫检测工作,我们还分别在E.coli中表达与纯化了重组蛋白Ag85A、ESAT-6以及嵌合抗原Ag856A2,并同时制备了Anti-Ag85A及Anti-ESAT-6的小鼠单克隆抗体。接下来我们的工作重点将是进行小鼠攻击试验,以评估嵌合基因重组卡介苗的免疫保护效果,为日后的临床试验打下基础。
[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
【学位授予单位】：复旦大学
【学位级别】：博士
【学位授予年份】：2008
【分类号】：R392
本文编号：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
【学位授予单位】：复旦大学
【学位级别】：博士
【学位授予年份】：2008
【分类号】：R392
【引证文献】
相关硕士学位论文 前2条
1 孙青;结核分枝杆菌无毒株H37Ra启动子突变基因的比较分析[D];苏州大学;2010年
2 尹文东;结核分枝杆菌重组Ag85A、Ag85B蛋白联合母牛分枝杆菌免疫原性的研究[D];吉林大学;2012年
，本文编号：2503023
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