响应群体感应和体内信号的细菌调控系统的设计及应用

发布时间：2017-12-31 18:43

本文关键词：响应群体感应和体内信号的细菌调控系统的设计及应用　出处：《华东理工大学》2015年博士论文　论文类型：学位论文

【摘要】：细菌载体疫苗因其具有天然的侵染能力、可携带多价抗原、具有佐剂功能以及能引起持久的黏膜免疫等特性,已成为当前疫苗研究领域的热点。维持较高的抗原表达效率和生物安全性对细菌载体疫苗的设计至关重要。为了提高抗原表达、降低代谢负担,采用体内诱导表达系统是一个较好的解决方案。本课题将费氏弧菌的群体感应基因与多种体内诱导调控元件进行整合,使群体感应基因能够感受宿主体内信号而被诱导开启,成为具有表达量高、易于调控、广宿主等优点的抗原表达系统。该系统不仅在模拟宿主体内的培养环境中功能良好,更在巨噬细胞、斑马鱼幼鱼及成鱼等体内系统中表现出优良的调控表达特性。将上述多元调控表达系统转入减毒迟缓爱德华氏菌中构建多价细菌载体疫苗,发现在体外培养环境中,含有该系统的载体疫苗能够正常生长,保持较高的活性和侵染能力；在宿主体内环境中,疫苗菌可以响应体内信号及细菌密度信号而开启表达抗原蛋白,并表现出较好的免疫保护力。另外,为了提高疫苗的生物安全性,本课题将群体感应基因和体内限铁诱导调控元件分别与裂解基因进行整合,在构建诱导裂解系统方面进行了探索性研究。首先,以费氏弧菌的Ⅰ型群体感应基因luxI-luxR作为基础,设计并筛选出了具有较高表达量的群体感应表达系统。该系统受到细菌密度的严格调控：当持续低密度培养时,系统表达量很低；而当菌体达到密度阈值后,系统迅速开启并高效表达。该系统具有表达效率高、调控严谨以及广泛的宿主适应性等优点。其次,根据体内缺乏游离铁离子和阿拉伯糖的信号特征,在上述群体感应表达系统的基础上偶联体内调控元件,构建了受体内信号和群体密度双信号调控的蛋白表达系统：(1)将群体感应系统与感受体内限铁信号的基因元件(如限铁启动子PviuA或铁摄取蛋白结合位点Fur box)相互偶联,构建了受“铁-细菌密度”双重信号调控的表达系统；(2)将群体感应系统与感应阿拉伯糖浓度的阿拉伯糖启动子ParaBAD以及群体淬灭基因aiiA相耦合,构建了受“阿拉伯糖-细菌密度”双重信号调控的体内诱导表达系统。在体外培养时,两个表达系统均能够严谨关闭,当模拟体内环境(限铁或者限阿拉伯糖)并达到浓度阈值时,两个表达系统均能有效开启群体感应回路,进行外源蛋白表达。体内环境中,搭载报告基因katushka的上述双信号调控表达系统在侵染J774巨噬细胞和斑马鱼幼鱼后均有明显的红色荧光表达,且随着菌体增殖,信号逐渐增强,体现出了良好的双信号调控特性。将编码嗜水气单胞菌(Aeromonas hydrophila) LSA34的GAPDH编码基因引入两个系统作为保护性抗原,转入减毒疫苗迟缓爱德华氏菌WED宿主中构建成多价细菌载体疫苗,将候选疫苗在大菱鲆(Scophtalmus maximus)中进行免疫保护力评价,结果显示接种这两种载体疫苗的大菱鲆对迟缓爱德华氏菌和嗜水气单胞菌野生株的侵染具有明显的抵御能力。说明这两个体内诱导表达系统在细菌载体疫苗设计中得到了很好地应用。最后,本课题采取体内诱导裂解的策略以提高细菌载体疫苗的生物安全性,在构建体内诱导裂解系统方面进行了探索性研究。将群体感应回路以及限铁诱导基因元件分别与裂解基因进行整合,构建了能够感应限铁信号而开启的体内诱导裂解系统。该系统能在体外正常培养时保持关闭,而在限铁环境和体内环境中表现出良好的裂解效果。含有该系统的鳗弧菌(Vibrio anguillarum)野生株MVM425在侵染斑马鱼(Danio rerio)后能够感受到宿主体内的限铁信号而有效裂解,从而使宿主得以存活。并且,将该系统转入鳗弧菌减毒疫苗株MVAV6203,不仅维持了其免疫保护力,而且消除了过量免疫对鱼体的侵害,极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.

【学位授予单位】：华东理工大学
【学位级别】：博士
【学位授予年份】：2015
【分类号】：Q93

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