基于体内诱导表达系统在迟钝爱德华氏菌中构建多价载体疫苗

发布时间：2018-04-18 23:24

本文选题：铁调控启动子 + 迟钝爱德华氏菌　；参考：《华东理工大学》2011年硕士论文

【摘要】：重组细菌载体疫苗作为一种有效的策略可以携带抗原蛋白并激发宿主的产生多重免疫保护力。目前,利用细菌载体活疫苗作为递呈载体是新兴现代疫苗的重要方向,接种这种疫苗后,机体既可产生针对载体细菌来源的病原的免疫保护力,又可产生针对携带抗原来源病原的免疫保护力,能够达到同时预防多种病原感染的作用。然而,活菌载体的安全性、外源基因表达稳定性等问题也是细菌载体疫苗发展中不可忽视的问题。迟钝爱德华氏菌(Edwardsiella tarda)是一种重要的革兰氏阴性鱼类病原菌。该病原菌能引起鱼类的出血性败血症,在本实验室前期工作中,已经从发病大菱鲆体内分离得到一株迟钝爱德华氏菌EIB202强毒株,并对其进行了全基因组测序,并构建了一系列适用于载体活菌疫苗的缺失减毒菌株。在本研究中,我们筛选了53个不同细菌来源的铁调控启动子,并检测了它们在体外培养时的转录活性。将筛选得到的具有较好铁调控活性的启动子Pdps和Pdps在巨噬细胞内进行胞内转录活性分析,最终选取了能够在胞内高效表达的启动子Pdps,并在模式动物斑马鱼上进行了观察检测。实验证明Pdps在体内有一个转录增强的趋势,可以用于体内诱导表达系统的构建。为了证明该系统能够作为成熟的抗原表达系统,我们将来源于嗜水气单胞菌(Aeromonas hydrophila) LSA34中的保护性抗原GAPDH连入含有Pdps的重组质粒中,同时,选取迟钝爱德华氏菌的减毒株WED作为宿主菌,构建了细菌载体疫苗WED/pUTDgap.大菱鲆免疫攻毒实验证明,WED/pUTDgap针对对野生型迟钝爱德华氏菌和嗜水气单胞菌均有超过60%的免疫保护力。本研究在迟钝爱德华氏菌中成功建立了基于铁诱导启动子的体内诱导抗原表达系统,并在迟钝爱德华氏菌减毒株WED中实现了嗜水气单胞菌保护性抗原蛋白的体内表达,成功构建了一株具有二价免疫保护力的迟钝爱德华氏菌减毒活疫苗候选株。
[Abstract]:As an effective strategy, recombinant bacterial vector vaccine can carry antigen protein and stimulate multiple immune protection of host.At present, it is an important direction of the new modern vaccine to use the live vaccine of bacterial carrier as the delivery vector. After inoculating this vaccine, the body can not only produce the immune protection ability against the pathogen from which the carrier bacteria come from.It can also produce immune protection against pathogens carrying antigens, and can prevent many kinds of pathogens at the same time.However, the safety of live bacterial vectors and the stability of foreign gene expression are also the problems that can not be ignored in the development of bacterial vector vaccine.Edwardsiella tarda is an important gram-negative fish pathogen.The pathogen can cause hemorrhagic septicemia in fish. In our laboratory, a virulent strain of Euphorbia obtuse (EIB202) was isolated from the infected turbot and sequenced.A series of deletion attenuated strains suitable for vector live vaccine were constructed.In this study, we screened 53 iron regulatory promoters from different bacterial sources and measured their transcriptional activities in vitro.The Pdps and Pdps promoters with better iron regulatory activity were selected for intracellular transcriptional activity analysis in macrophages, and Pdpss, which were highly expressed in the cells, were selected and detected on zebrafish, a model animal.The results show that Pdps has a tendency of transcriptional enhancement in vivo and can be used to construct an in vivo induced expression system.In order to prove that the system can be used as a mature antigen expression system, we linked the protective antigen GAPDH from Aeromonas hydrophila LSA34 to the recombinant plasmid containing Pdps.The vector vaccine WED- pUTDgap. was constructed by selecting the attenuated strain WED of Edwardsiella obtuse as the host strain.The results showed that WED- pUTDgap had more than 60% protection against both wild type Edwardiella obtuse and Aeromonas hydrophila.In this study, we successfully established an in vivo antigen expression system based on iron induced promoters in Edouard dullus, and realized the expression of protective antigen protein of Aeromonas hydrophila in WED, a attenuated strain of E. dullus.A bivalent vaccine candidate for attenuated live vaccine of Edwardsiella dullus was successfully constructed.
【学位授予单位】：华东理工大学
【学位级别】：硕士
【学位授予年份】：2011
【分类号】：R392.1

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