迟缓爱德华氏菌基因工程疫苗的构建及免疫效果的研究

发布时间：2018-05-12 06:50

  本文选题：迟缓爱德华氏菌 + 保护性抗原　； 参考：《中国科学院研究生院（海洋研究所）》2010年博士论文

【摘要】： 迟缓爱德华氏菌(Edwardsiella tarda)是一种鱼类病原菌,其感染鱼类后引起迟缓爱德华菌病(edwardsiellosis),给养殖业造成严重的经济损失。 本研究中,应用IVIAT技术,我们从病鱼体内分离的迟缓爱德华氏菌菌株TX01中筛选得到Eta21、Eta6和FliC三个抗原蛋白。其中Eta21与其他菌株中一个潜在的多肽酶相似性较高;Eta6与大肠杆菌中的Ecotin前体蛋白同源;FliC是一种鞭毛蛋白。以牙鲆(Japanese flounder)作为模式动物的研究中,发现重组Eta21蛋白能够有效的保护牙鲆抵抗迟缓爱德华氏菌的侵染;重组Eta6蛋白具有一定的保护效应;重组FliC蛋白没有明显的保护效应。 以eta6和fliC基因构建的DNA疫苗pETA6和pFliC的相对保护效率分别为50%和33%。与pETA6的保护效应相比,将eta6基因和fliC基因连在一起构建的融合DNA疫苗pCE6的保护效应显著提高。同时,将前期鉴定出的迟缓爱德华氏菌TX01的抗原基因et18连接到fliC基因的后面,构建了融合DNA疫苗pCE18。与能单纯表达Et18的DNA疫苗pEt18相比,pCE18免疫牙鲆后可以产生更好的保护效应。pEta6和pCE6免疫后,牙鲆体内产生了特异性的抗体,而且其体内一些与固有免疫和获得性免疫相关基因的表达水平也获得了提高。相对于pEta6,pCE6诱导的上述基因表达水平提高的幅度更为明显。 为提高Eta21的保护效应,我们将eta21基因与胞外琼胶酶agaV的分泌片段基因融合,构建了重组质粒pTAET21。以含有此重组质粒的大肠杆菌DH5α作为活菌疫苗,其保护效应较之重组蛋白Eta21的保护能力有明显的提高。
[Abstract]:Edwardsiella tardaa is a kind of fish pathogen, which causes Edwardsiella tarda disease after it is infected with fish, and causes serious economic loss to the aquaculture industry. In this study, three antigenic proteins, Eta21, Eta6 and FliC, were isolated from the Eta21 Eta6 and FliC strains of Edwardian tardy strain TX01 by using IVIAT technique. There is a high similarity between Eta21 and a potential polypeptidase in other strains. Eta6 is a flagellin with Ecotin precursor protein homologous in Escherichia coli. Using Japanese flounder as a model animal, it was found that the recombinant Eta21 protein could effectively protect Paralichthys olivaceus from infection by Edwarder, the recombinant Eta6 protein had some protective effect, and the recombinant FliC protein had no obvious protective effect. The relative protective efficiency of DNA vaccine pETA6 and pFliC constructed by eta6 and fliC gene were 50% and 33% respectively. Compared with the protective effect of pETA6, the protective effect of the fusion DNA vaccine pCE6 constructed by linking eta6 gene and fliC gene was significantly improved. At the same time, the previously identified TX01 antigen gene et18 was linked to the fliC gene, and the fusion DNA vaccine pCE18 was constructed. Compared with the DNA vaccine pEt18, which can express Et18 alone, pCE18 can produce better protective effect after immunization. PEta6 and pCE6 can produce specific antibody in flounder. Moreover, the expression level of some genes related to innate immunity and acquired immunity were also improved. Compared with pEta6, pCE6 induced gene expression increased more significantly. In order to improve the protective effect of Eta21, we fused the eta21 gene with the secreting fragment of extracellular agarose enzyme agaV and constructed the recombinant plasmid pTAET21. The protective effect of Escherichia coli DH5 伪 containing the recombinant plasmid as a live vaccine was significantly higher than that of the recombinant protein Eta21.
【学位授予单位】：中国科学院研究生院（海洋研究所）
【学位级别】：博士
【学位授予年份】：2010
【分类号】：R392.1

【参考文献】

相关期刊论文 前5条

1 郭国强;陈万光;郭黛健;;鱼类疫苗的选择和使用[J];科学养鱼;2007年11期

2 刘云,姜国良,姜明,张士璀;牙鲆鳃淋巴样组织内免疫相关细胞的超微结构[J];青岛海洋大学学报(自然科学版);2001年06期

3 侯进慧;袁军;;鱼用基因工程疫苗[J];生命的化学;2009年03期

4 王涛;刘纯杰;;反向疫苗学分析方法及在疫苗开发中应用进展[J];微生物学免疫学进展;2006年01期

5 单晓枫,高云航,李影,钱爱东;鱼用疫苗研究进展[J];中国兽药杂志;2005年11期

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