本文選題：炭疽芽孢桿菌　切入點：芽孢　出處：《中國人民解放軍軍事醫(yī)學(xué)科學(xué)院》2009年博士論文　論文類型：學(xué)位論文

【摘要】： 炭疽芽孢桿菌(Bacillus anthracis)是一種G+菌,它引起的炭疽是一種人獸共患的烈性傳染病,嚴(yán)重危害人類的健康,在我國列為乙類傳染病。同時它又是一種潛在的生物戰(zhàn)劑。因此,炭疽疫苗一直是生物安全研究的一個重點,而且炭疽疫苗的研究也一直是病原微生物及其疫苗研究領(lǐng)域的一個重點。炭疽桿菌AP422(pXO1-pXO2-)是一株不含有兩個毒性大質(zhì)粒,但又能夠形成芽孢的減毒菌株,具有潛在的應(yīng)用前景。本研究的工作也正是從AP422入手,分別采用基因敲除和基因敲入兩個不同的方式,對該菌株作進一步的基因改造和分析評價,以期得到一種新型的炭疽芽孢疫苗載體,為炭疽疫苗研究奠定良好的實驗基礎(chǔ)。 在第一部分工作中,以炭疽芽孢桿菌S-層蛋白EA1的編碼基因eag為目標(biāo),通過一系列實驗建立了Cre-LoxP系統(tǒng)在炭疽芽孢桿菌中進行基因打靶的技術(shù)方法。確定了實驗過程中的幾個關(guān)鍵點,如打靶質(zhì)粒、同源臂長度、高效電轉(zhuǎn)化方法、抗生素濃度和篩選培養(yǎng)方式等。這樣,通過對不同條件的考查和實驗分析,最終建立了高效的基因打靶的方法。在此基礎(chǔ)上我們成功地對S-層蛋白EA1的編碼基因eag進行了敲除,并在基因組水平、RNA水平和蛋白質(zhì)水平等多個層次進行了確證。與此同時,通過對eag基因敲除株的蛋白表達(dá)情況的初步分析,我們發(fā)現(xiàn),S-層蛋白的合成可能存在一種代償機制,當(dāng)天然狀態(tài)下主要表達(dá)的S-層蛋白不再表達(dá)時,其它相關(guān)的S-層蛋白的表達(dá)水平明顯上調(diào),以保證S-層的完整性。 在第二部分工作中,為避免重組菌株對抗生素選擇壓力的依賴,建立平衡致死系統(tǒng),通過基因敲除的方式構(gòu)建了胸腺嘧啶營養(yǎng)缺陷的炭疽桿菌,并對其表型進行了綜合分析。具體而言,我們通過生物信息學(xué)分析獲得在炭疽芽孢桿菌中負(fù)責(zé)胸腺嘧啶合成的關(guān)鍵基因,選定胸腺嘧啶合成的關(guān)鍵酶即胸腺嘧啶合成酶基因thyA和核黃素依賴的胸腺嘧啶合成酶基因thyX作為我們進行基因敲除的目標(biāo)。利用同源重組原理,成功敲除炭疽桿菌中胸腺嘧啶合成的2個關(guān)鍵基因,并且結(jié)合Cre-LoxP系統(tǒng)的作用,得到不含抗性標(biāo)記的重組菌B. anthracis AP422ΔthyAΔthyX。表型分析證明該菌株的生長狀態(tài)表現(xiàn)為胸腺嘧啶營養(yǎng)依賴,同時,缺陷菌株形成芽孢的能力與AP422相比,不但沒有降低,反而在一定程度上有一定的增強。這樣,通過營養(yǎng)缺陷型的改造,菌株AP422ΔthyAΔthyX就有可能發(fā)展成一種平衡致死系統(tǒng)用的新的表達(dá)宿主。 通過對單獨敲除thyA基因和thyX的菌株進行分析表明,在炭疽桿菌中,可能存在兩種不同的胸腺嘧啶合成的代謝通路,即TS/DHFR通路和FDTS通路。這兩條通路可以獨立發(fā)揮作用,都可以保證菌株的正常生長。我們利用λRed系統(tǒng),同樣利用基因敲除的方法得到了胸腺嘧啶營養(yǎng)缺陷的大腸桿菌,并以其為宿主菌,對炭疽桿菌的thyA和thyX基因及其產(chǎn)物的生物學(xué)功能進行了初步的確證。結(jié)果表明,在E.coli中,二者都能夠發(fā)揮胸腺嘧啶合成酶的功能,使相應(yīng)的營養(yǎng)缺陷菌株恢復(fù)正常的生長。另外,我們的研究也表明,thyX基因產(chǎn)物FDTS的存在,對于炭疽桿菌的甲氧芐啶(二氫葉酸還原酶抑制劑)的抗藥性起一定的作用。 第三部分工作中,我們采用基因敲入的方法,建立了基于炭疽桿菌S層蛋白EA1的表面呈現(xiàn)體系,實現(xiàn)了炭疽疫苗相關(guān)抗原在不同炭疽桿菌表面的呈現(xiàn)表達(dá)。通過同源重組的方式,結(jié)合Cre-LoxP系統(tǒng),我們成功實現(xiàn)了在炭疽桿菌中基因的“無痕”敲入。具體而言,以EA1的保守結(jié)構(gòu)域SLH結(jié)構(gòu)域為錨定部分,在炭疽S層表面成功展示了PA20、PAⅣ和LFb三個抗原結(jié)構(gòu)域。蛋白水平分析表明,目標(biāo)蛋白的確以融合蛋白的形式表達(dá);免疫熒光實驗證明,目標(biāo)蛋白融合蛋白的形式表達(dá)呈現(xiàn)于菌體表面;芽孢形成實驗證實,重組菌仍具有形成芽孢的能力;而且免疫學(xué)實驗也表明,無論是經(jīng)以菌體的形式進行免疫,還是以芽孢的形式進行免疫,都能激發(fā)實驗動物有效的免疫應(yīng)答。以菌體形式鼻飼免疫時,三種重組菌都能夠激發(fā)豚鼠很好的免疫應(yīng)答,血清IgG滴度均在1:1000以上。而且不同劑量的呈現(xiàn)PAⅣ的重組菌以芽孢的形式進行口服免疫豚鼠時,同樣能激發(fā)有效的免疫應(yīng)答。不但血清IgG水平在1:1000左右,而且也能夠檢測到抗原特異性的分泌型IgA的產(chǎn)生。與此同時,以芽孢進行免疫時,同樣能激發(fā)豚鼠對于芽孢蛋白的免疫應(yīng)答,這對于提高芽孢的免疫保護效果有著十分重要的意義。另外,實驗中得到的重組菌不帶有任何抗性標(biāo)記,符合當(dāng)今對活載體疫苗的要求。這一部分的研究也充分表明,AP422作為一種減毒的炭疽桿菌,可以作為一種炭疽桿菌芽孢疫苗載體的候選菌株。 在第四部分工作中,利用能在芽孢蛋白BclA上呈現(xiàn)LFb的重組菌,實現(xiàn)了PAⅣ和LFb的共同呈現(xiàn)表達(dá)。由于LFb的表達(dá)也能呈現(xiàn)在芽孢表面,以芽孢形式免疫后能直接發(fā)揮作用,芽孢萌發(fā)后則通過表達(dá)PAⅣ來發(fā)揮作用,從而達(dá)到二價疫苗的效果。免疫學(xué)評價表明,無論是呈現(xiàn)于芽孢表面的LFb,還是呈現(xiàn)于S-層的PAⅣ,都能夠激發(fā)豚鼠顯著的免疫應(yīng)答免疫應(yīng)答。 總之,我們的研究表明,炭疽桿菌AP422可以發(fā)展為一種良好的炭疽桿菌芽孢疫苗載體。
[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.

【學(xué)位授予單位】：中國人民解放軍軍事醫(yī)學(xué)科學(xué)院
【學(xué)位級別】：博士
【學(xué)位授予年份】：2009
【分類號】：R392.1

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