本文關(guān)鍵詞：響應(yīng)群體感應(yīng)和體內(nèi)信號的細(xì)菌調(diào)控系統(tǒng)的設(shè)計及應(yīng)用　出處：《華東理工大學(xué)》2015年博士論文　論文類型：學(xué)位論文

  更多相關(guān)文章： 細(xì)菌載體疫苗 體內(nèi)誘導(dǎo)表達(dá) 體內(nèi)誘導(dǎo)裂解 群體感應(yīng)系統(tǒng)

【摘要】：細(xì)菌載體疫苗因其具有天然的侵染能力、可攜帶多價抗原、具有佐劑功能以及能引起持久的黏膜免疫等特性,已成為當(dāng)前疫苗研究領(lǐng)域的熱點(diǎn)。維持較高的抗原表達(dá)效率和生物安全性對細(xì)菌載體疫苗的設(shè)計至關(guān)重要。為了提高抗原表達(dá)、降低代謝負(fù)擔(dān),采用體內(nèi)誘導(dǎo)表達(dá)系統(tǒng)是一個較好的解決方案。本課題將費(fèi)氏弧菌的群體感應(yīng)基因與多種體內(nèi)誘導(dǎo)調(diào)控元件進(jìn)行整合,使群體感應(yīng)基因能夠感受宿主體內(nèi)信號而被誘導(dǎo)開啟,成為具有表達(dá)量高、易于調(diào)控、廣宿主等優(yōu)點(diǎn)的抗原表達(dá)系統(tǒng)。該系統(tǒng)不僅在模擬宿主體內(nèi)的培養(yǎng)環(huán)境中功能良好,更在巨噬細(xì)胞、斑馬魚幼魚及成魚等體內(nèi)系統(tǒng)中表現(xiàn)出優(yōu)良的調(diào)控表達(dá)特性。將上述多元調(diào)控表達(dá)系統(tǒng)轉(zhuǎn)入減毒遲緩愛德華氏菌中構(gòu)建多價細(xì)菌載體疫苗,發(fā)現(xiàn)在體外培養(yǎng)環(huán)境中,含有該系統(tǒng)的載體疫苗能夠正常生長,保持較高的活性和侵染能力；在宿主體內(nèi)環(huán)境中,疫苗菌可以響應(yīng)體內(nèi)信號及細(xì)菌密度信號而開啟表達(dá)抗原蛋白,并表現(xiàn)出較好的免疫保護(hù)力。另外,為了提高疫苗的生物安全性,本課題將群體感應(yīng)基因和體內(nèi)限鐵誘導(dǎo)調(diào)控元件分別與裂解基因進(jìn)行整合,在構(gòu)建誘導(dǎo)裂解系統(tǒng)方面進(jìn)行了探索性研究。首先,以費(fèi)氏弧菌的Ⅰ型群體感應(yīng)基因luxI-luxR作為基礎(chǔ),設(shè)計并篩選出了具有較高表達(dá)量的群體感應(yīng)表達(dá)系統(tǒng)。該系統(tǒng)受到細(xì)菌密度的嚴(yán)格調(diào)控：當(dāng)持續(xù)低密度培養(yǎng)時,系統(tǒng)表達(dá)量很低；而當(dāng)菌體達(dá)到密度閾值后,系統(tǒng)迅速開啟并高效表達(dá)。該系統(tǒng)具有表達(dá)效率高、調(diào)控嚴(yán)謹(jǐn)以及廣泛的宿主適應(yīng)性等優(yōu)點(diǎn)。其次,根據(jù)體內(nèi)缺乏游離鐵離子和阿拉伯糖的信號特征,在上述群體感應(yīng)表達(dá)系統(tǒng)的基礎(chǔ)上偶聯(lián)體內(nèi)調(diào)控元件,構(gòu)建了受體內(nèi)信號和群體密度雙信號調(diào)控的蛋白表達(dá)系統(tǒng)：(1)將群體感應(yīng)系統(tǒng)與感受體內(nèi)限鐵信號的基因元件(如限鐵啟動子PviuA或鐵攝取蛋白結(jié)合位點(diǎn)Fur box)相互偶聯(lián),構(gòu)建了受“鐵-細(xì)菌密度”雙重信號調(diào)控的表達(dá)系統(tǒng)；(2)將群體感應(yīng)系統(tǒng)與感應(yīng)阿拉伯糖濃度的阿拉伯糖啟動子ParaBAD以及群體淬滅基因aiiA相耦合,構(gòu)建了受“阿拉伯糖-細(xì)菌密度”雙重信號調(diào)控的體內(nèi)誘導(dǎo)表達(dá)系統(tǒng)。在體外培養(yǎng)時,兩個表達(dá)系統(tǒng)均能夠嚴(yán)謹(jǐn)關(guān)閉,當(dāng)模擬體內(nèi)環(huán)境(限鐵或者限阿拉伯糖)并達(dá)到濃度閾值時,兩個表達(dá)系統(tǒng)均能有效開啟群體感應(yīng)回路,進(jìn)行外源蛋白表達(dá)。體內(nèi)環(huán)境中,搭載報告基因katushka的上述雙信號調(diào)控表達(dá)系統(tǒng)在侵染J774巨噬細(xì)胞和斑馬魚幼魚后均有明顯的紅色熒光表達(dá),且隨著菌體增殖,信號逐漸增強(qiáng),體現(xiàn)出了良好的雙信號調(diào)控特性。將編碼嗜水氣單胞菌(Aeromonas hydrophila) LSA34的GAPDH編碼基因引入兩個系統(tǒng)作為保護(hù)性抗原,轉(zhuǎn)入減毒疫苗遲緩愛德華氏菌WED宿主中構(gòu)建成多價細(xì)菌載體疫苗,將候選疫苗在大菱鲆(Scophtalmus maximus)中進(jìn)行免疫保護(hù)力評價,結(jié)果顯示接種這兩種載體疫苗的大菱鲆對遲緩愛德華氏菌和嗜水氣單胞菌野生株的侵染具有明顯的抵御能力。說明這兩個體內(nèi)誘導(dǎo)表達(dá)系統(tǒng)在細(xì)菌載體疫苗設(shè)計中得到了很好地應(yīng)用。最后,本課題采取體內(nèi)誘導(dǎo)裂解的策略以提高細(xì)菌載體疫苗的生物安全性,在構(gòu)建體內(nèi)誘導(dǎo)裂解系統(tǒng)方面進(jìn)行了探索性研究。將群體感應(yīng)回路以及限鐵誘導(dǎo)基因元件分別與裂解基因進(jìn)行整合,構(gòu)建了能夠感應(yīng)限鐵信號而開啟的體內(nèi)誘導(dǎo)裂解系統(tǒng)。該系統(tǒng)能在體外正常培養(yǎng)時保持關(guān)閉,而在限鐵環(huán)境和體內(nèi)環(huán)境中表現(xiàn)出良好的裂解效果。含有該系統(tǒng)的鰻弧菌(Vibrio anguillarum)野生株MVM425在侵染斑馬魚(Danio rerio)后能夠感受到宿主體內(nèi)的限鐵信號而有效裂解,從而使宿主得以存活。并且,將該系統(tǒng)轉(zhuǎn)入鰻弧菌減毒疫苗株MVAV6203,不僅維持了其免疫保護(hù)力,而且消除了過量免疫對魚體的侵害,極大提升了其生物安全性。
[Abstract]:Bacterial vector vaccine because of its natural infection ability, can carry a multivalent antigen, with adjuvant and can cause persistent mucosal immunity and other characteristics, has become a hotspot in the field of vaccine research. To maintain a high efficiency of antigen expression and biological safety is crucial to the design of bacterial vector vaccine. In order to improve the expression of reduced metabolic burden and by in vivo induced gene expression system is a good solution. The Vibrio fischeri quorum sensing gene and in vivo induced regulatory element integration, the quorum sensing gene can feel the host signal is induced into open, with high expression amount, easy to control, the advantages of broad host antigen expression system the system not only in the culture environment simulation host in good function in macrophages, zebrafish larvae and adult fish in vivo system Express excellent regulation. The regulation of multiple expression system to construct a multivalent bacterial vector vaccine by Edwardsiella tarda, found in vitro, vector vaccine containing the system can maintain the normal growth, high activity and infection ability; in the host environment, vaccine strain can respond in vivo signals and the density of bacteria signal and open expression of antigen, and showed good protective immunity. In addition, in order to improve the biological safety of the vaccine, the quorum sensing gene and in vivo induced limited iron regulatory elements were integrated with lytic gene was studied in the construction of system induced cleavage. First of all, to type quorum sensing gene luxI-luxR of Vibrio fischeri as the foundation, designed and screened with high expression of quorum sensing expression system. This system Strictly controlled by the bacterial density: when the continuous low density culture system, the expression is very low; and when the cell density reached the threshold, the system quickly opened and highly expressed. The system has high expression efficiency, strict regulation and wide host adaptability. Secondly, according to the signal characteristics in the lack of free iron ions and Arabia sugar, based on the quorum sensing system on the expression of couplet in regulatory elements, built by the in vivo signal and the population density of double signal regulated protein expression system: (1) the quorum sensing system and feel the limit genetic element signals (such as iron limited iron promoter PviuA or iron uptake protein binding sites Fur box) the mutual coupling, construct the expression system by the "iron bacteria density" dual signal control; (2) the quorum sensing system and induction of Arabia sugar concentration of Arabia sugar promoter ParaB AD and the quenching of aiiA gene coupled to construct the expression system induced by "Arabia sugar - bacterial density" double signal regulation in vivo. In vitro, two expression systems were able to close the rigorous, simulating the in vivo environment when (the iron or Arabia sugar) and reached the concentration threshold, two the expression system can effectively open the quorum sensing loop, exogenous protein expression. In vivo, the dual signal control with katushka reporter gene expression system in J774 infected macrophages and zebrafish larvae were significantly red fluorescent expression with cell proliferation, signal gradually increased, reflecting the good characteristics of the double signal control the encoding of Aeromonas hydrophila (Aeromonas hydrophila) GAPDH gene encoding LSA34 into two systems as protective antigen into the attenuated vaccine strain WED delayed Edward's host To construct a multivalent bacterial vector vaccine, the vaccine candidate in turbot (Scophtalmus maximus) in the protective immunity evaluation, the results showed that inoculation with the two kinds of vaccine of turbot of Edwardsiella tarda and Aeromonas hydrophila infection of wild strains has obvious ability to resist. This shows two in vivo induced expression system has been well applied in bacterial vector vaccine design. Finally, this paper adopts the in vivo induced cleavage of the strategies to improve the biological safety of bacterial vector vaccine, was studied in the construction of in vivo induced cracking system. The quorum sensing circuit and the limit of iron induced gene element respectively and lytic genes were integrated to construct to open the iron induction limit signal in vivo induced cracking system. The system can maintain normal closed in vitro culture, and in the iron environment and in vivo environment Show cracking good effect. The system contains the vibrioanguillarum (Vibrio anguillarum) wild strain MVM425 (Danio rerio) infection in the zebrafish can feel iron limited signal host and effective cracking, so that the host to survive. Also, the system into the attenuated Vibrio anguillarum strain MVAV6203, not only to maintain the protective immunity, but also eliminate the harmful immune to the fish body, greatly enhance the biological safety.

【學(xué)位授予單位】：華東理工大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2015
【分類號】：Q93

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本文編號：1361022