炭疽杆菌芽孢疫苗载体的探索性研究

发布时间：2018-03-14 23:06

本文选题：炭疽芽孢杆菌　切入点：芽孢　出处：《中国人民解放军军事医学科学院》2009年博士论文　论文类型：学位论文

【摘要】： 炭疽芽孢杆菌(Bacillus anthracis)是一种G+菌,它引起的炭疽是一种人兽共患的烈性传染病,严重危害人类的健康,在我国列为乙类传染病。同时它又是一种潜在的生物战剂。因此,炭疽疫苗一直是生物安全研究的一个重点,而且炭疽疫苗的研究也一直是病原微生物及其疫苗研究领域的一个重点。炭疽杆菌AP422(pXO1-pXO2-)是一株不含有两个毒性大质粒,但又能够形成芽孢的减毒菌株,具有潜在的应用前景。本研究的工作也正是从AP422入手,分别采用基因敲除和基因敲入两个不同的方式,对该菌株作进一步的基因改造和分析评价,以期得到一种新型的炭疽芽孢疫苗载体,为炭疽疫苗研究奠定良好的实验基础。在第一部分工作中,以炭疽芽孢杆菌S-层蛋白EA1的编码基因eag为目标,通过一系列实验建立了Cre-LoxP系统在炭疽芽孢杆菌中进行基因打靶的技术方法。确定了实验过程中的几个关键点,如打靶质粒、同源臂长度、高效电转化方法、抗生素浓度和筛选培养方式等。这样,通过对不同条件的考查和实验分析,最终建立了高效的基因打靶的方法。在此基础上我们成功地对S-层蛋白EA1的编码基因eag进行了敲除,并在基因组水平、RNA水平和蛋白质水平等多个层次进行了确证。与此同时,通过对eag基因敲除株的蛋白表达情况的初步分析,我们发现,S-层蛋白的合成可能存在一种代偿机制,当天然状态下主要表达的S-层蛋白不再表达时,其它相关的S-层蛋白的表达水平明显上调,以保证S-层的完整性。在第二部分工作中,为避免重组菌株对抗生素选择压力的依赖,建立平衡致死系统,通过基因敲除的方式构建了胸腺嘧啶营养缺陷的炭疽杆菌,并对其表型进行了综合分析。具体而言,我们通过生物信息学分析获得在炭疽芽孢杆菌中负责胸腺嘧啶合成的关键基因,选定胸腺嘧啶合成的关键酶即胸腺嘧啶合成酶基因thyA和核黄素依赖的胸腺嘧啶合成酶基因thyX作为我们进行基因敲除的目标。利用同源重组原理,成功敲除炭疽杆菌中胸腺嘧啶合成的2个关键基因,并且结合Cre-LoxP系统的作用,得到不含抗性标记的重组菌B. anthracis AP422ΔthyAΔthyX。表型分析证明该菌株的生长状态表现为胸腺嘧啶营养依赖,同时,缺陷菌株形成芽孢的能力与AP422相比,不但没有降低,反而在一定程度上有一定的增强。这样,通过营养缺陷型的改造,菌株AP422ΔthyAΔthyX就有可能发展成一种平衡致死系统用的新的表达宿主。通过对单独敲除thyA基因和thyX的菌株进行分析表明,在炭疽杆菌中,可能存在两种不同的胸腺嘧啶合成的代谢通路,即TS/DHFR通路和FDTS通路。这两条通路可以独立发挥作用,都可以保证菌株的正常生长。我们利用λRed系统,同样利用基因敲除的方法得到了胸腺嘧啶营养缺陷的大肠杆菌,并以其为宿主菌,对炭疽杆菌的thyA和thyX基因及其产物的生物学功能进行了初步的确证。结果表明,在E.coli中,二者都能够发挥胸腺嘧啶合成酶的功能,使相应的营养缺陷菌株恢复正常的生长。另外,我们的研究也表明,thyX基因产物FDTS的存在,对于炭疽杆菌的甲氧苄啶(二氢叶酸还原酶抑制剂)的抗药性起一定的作用。第三部分工作中,我们采用基因敲入的方法,建立了基于炭疽杆菌S层蛋白EA1的表面呈现体系,实现了炭疽疫苗相关抗原在不同炭疽杆菌表面的呈现表达。通过同源重组的方式,结合Cre-LoxP系统,我们成功实现了在炭疽杆菌中基因的“无痕”敲入。具体而言,以EA1的保守结构域SLH结构域为锚定部分,在炭疽S层表面成功展示了PA20、PAⅣ和LFb三个抗原结构域。蛋白水平分析表明,目标蛋白的确以融合蛋白的形式表达;免疫荧光实验证明,目标蛋白融合蛋白的形式表达呈现于菌体表面;芽孢形成实验证实,重组菌仍具有形成芽孢的能力;而且免疫学实验也表明,无论是经以菌体的形式进行免疫,还是以芽孢的形式进行免疫,都能激发实验动物有效的免疫应答。以菌体形式鼻饲免疫时,三种重组菌都能够激发豚鼠很好的免疫应答,血清IgG滴度均在1:1000以上。而且不同剂量的呈现PAⅣ的重组菌以芽孢的形式进行口服免疫豚鼠时,同样能激发有效的免疫应答。不但血清IgG水平在1:1000左右,而且也能够检测到抗原特异性的分泌型IgA的产生。与此同时,以芽孢进行免疫时,同样能激发豚鼠对于芽孢蛋白的免疫应答,这对于提高芽孢的免疫保护效果有着十分重要的意义。另外,实验中得到的重组菌不带有任何抗性标记,符合当今对活载体疫苗的要求。这一部分的研究也充分表明,AP422作为一种减毒的炭疽杆菌,可以作为一种炭疽杆菌芽孢疫苗载体的候选菌株。在第四部分工作中,利用能在芽孢蛋白BclA上呈现LFb的重组菌,实现了PAⅣ和LFb的共同呈现表达。由于LFb的表达也能呈现在芽孢表面,以芽孢形式免疫后能直接发挥作用,芽孢萌发后则通过表达PAⅣ来发挥作用,从而达到二价疫苗的效果。免疫学评价表明,无论是呈现于芽孢表面的LFb,还是呈现于S-层的PAⅣ,都能够激发豚鼠显著的免疫应答免疫应答。总之,我们的研究表明,炭疽杆菌AP422可以发展为一种良好的炭疽杆菌芽孢疫苗载体。
[Abstract]:Bacillus anthracis (Bacillus anthracis G+) is a kind of fungus, it causes anthrax is a zoonotic infectious disease, serious harm to human health, in our country listed as class B infectious diseases. It is also a potential biological warfare agent. Therefore, the anthrax vaccine has been a key biological the security research, and anthrax vaccine research has been a key pathogenic microorganism and its vaccine research field. Anthrax bacillus AP422 (pXO1-pXO2-) is a non toxic plasmid containing two, but also can form the attenuated strain of Bacillus, which has potential application prospect. This research work is from starting with AP422, respectively by gene knock-out and knock in two different ways, gene transformation and further analysis and evaluation of the strain, in order to get a new anthrax vaccine carrier for anthrax vaccine research has laid a good The basis of the experiment.
In the first part, the Bacillus anthracis S- layer EA1 protein encoding gene EAG as the target, technology and methods through a series of experiments to establish Cre-LoxP system for gene targeting in B.anthracis. Identified several key points in the process of experiments, such as targeting plasmid, homologous arm length, high power transformation methods. The concentration of antibiotics and screening methods. In this way, through the examination and analysis of different experimental conditions, the eventual establishment of efficient gene targeting methods. On this basis we successfully on S- layer EA1 protein encoding gene EAG was knocked out, and at the genomic level, multiple levels of RNA and protein levels. Were confirmed. At the same time, through to the knockout of EAG gene in the preliminary analysis, the protein expression strain we found that there may be a compensatory mechanism of synthesis of S- layer protein, the main natural state When the expression of S- layer protein is no longer expressed, the expression level of other related S- layer proteins is obviously up-regulated, in order to ensure the integrity of the S- layer.
In the second part, in order to avoid the dependence of the recombinant strain selection pressure of antibiotics, establish balanced lethal system by gene knockout method constructs thymidine auxotrophic anthrax, and the phenotype was analyzed. Specifically, we obtained by bioinformatics analysis of key genes responsible for the synthesis of thymidine in anthrax subtilis, selected key enzyme thymine synthesis that thymidylate synthase gene thyA and riboflavin dependent thymidylate synthase gene knockout of thyX as our goal. By using the principle of homologous recombination, successfully knocked out 2 key genes of Bacillus anthracis in the synthesis of thymine, and combined with the effect of the Cre-LoxP system, obtained without resistance marker recombinant B. anthracis AP422 Delta thyA Delta thyX. phenotype analysis demonstrated that the growth state of the strains showed its chest gland At the same time, ceftazidime nutrition dependence, mutant strains of spore forming ability compared with AP422, not only did not decrease, but there is an increase in a certain extent. So, through the transformation of auxotrophic strain AP422, Delta thyA Delta thyX is likely to develop into a balanced lethal system for the new expression of the host.
Based on the separate thyA gene knockout strains and thyX analysis showed that in Bacillus anthracis, there may be two different metabolic pathways of thymine synthesis, namely TS/DHFR and FDTS pathways. These two pathways can function independently, can ensure the normal growth of strains. We use a Red system using the same gene knockout method obtained the thymine auxotrophic Escherichia coli and its host bacteria, biological function of thyA and thyX genes of Bacillus anthracis and its products were preliminarily confirmed. The results showed that in E.coli, two of them are able to play the function of thymidylate synthase, the auxotrophic strains corresponding recovery normal growth. In addition, our study also showed that the thyX gene product FDTS, Bacillus anthracis for trimethoprim (dihydrofolate reductase inhibitors) resistance to certain The role.
In the third part, we use the method of gene knock in, a surface layer protein EA1 of Bacillus anthracis S rendering system based on the realization of the anthrax vaccine related antigens in different surface expression. Showing anthrax bacillus by homologous recombination. The combination of the Cre-LoxP system, we have achieved in the anthrax gene in "no trace" knockin. Specifically, the conservative domain of SLH EA1 for anchoring part in the anthrax S layer on the surface of the successful demonstration of PA20, PA IV and LFb three antigen domain. Protein level analysis showed that the target protein is expressed in the form of fusion protein; immunofluorescence assay proved the target protein, fusion protein the form of expression on the cell surface; spore formation experiments confirmed that the recombinant bacteria still has a spore forming ability; and immunological experiments also showed that both the immune cell to form, also Is immune to Bacillus form, can stimulate the immune response of experimental animal effectively. By cell immune form of nasal feeding, the immune response of three recombinant bacteria can stimulate the guinea pig good, serum IgG titer was above 1:1000. And different doses showed PA IV recombinant bacteria by oral immunization with spores of guinea pigs the form, also can stimulate immune response effectively. Not only the level of serum IgG in 1:1000, but also able to detect antigen-specific secretory IgA. At the same time, immune to bacillus, also can stimulate the immune response of guinea pigs to Bacillus protein, which is very important for improving the immune protective effect of Bacillus subtilis there is a significance. In addition, the recombinant strains did not carry any resistance marker, accord with the requirements of the vaccine. This part of the study also showed that AP422 as a The attenuated Bacillus anthracis can be used as a candidate for the Bacillus anthracis spore vaccine.
In the fourth part, the use of recombinant bacteria can render LFb in Bacillus protein BclA, to achieve the PA IV and LFb jointly presented expression. The expression of LFb can be found on the surface of spore, spore to form after immunization can directly play a role in spore germination after expression by PA IV to play a role, so to achieve the two valent vaccine effect. Immunological evaluation showed that both in spore surface LFb, or in S- layer of PA IV, can stimulate the immune response of guinea pig significant immune response.
In conclusion, our study shows that Bacillus anthracis AP422 can be developed as a good carrier of Bacillus anthracis spore vaccine.

【学位授予单位】：中国人民解放军军事医学科学院
【学位级别】：博士
【学位授予年份】：2009
【分类号】：R392.1

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