【作者】 王敏；

【导师】 常维山； 王雪鹏；

【作者基本信息】 山东农业大学 ， 预防兽医学， 2015， 硕士

【摘要】 迟缓爱德华菌是水产养殖业重要的致病菌,感染后很多鱼类出现全身性败血症、皮肤肿胀溃烂、内脏器官坏死、全身严重出血等症状;群体感应系统(Quorum sensing,QS)是种间交流的重要信号分子,是细菌种间交流的通用语言,自诱导物-2(Autoinducer-2,AI-2)的合成机制是革兰氏阳性菌与革兰氏阴性菌同时存在的唯一一个QS系统;AI-2的合成酶LuxS是AI-2合成的关键,同时也是甲基循环(Activated methyl cycle,AMC)的固有组分,Lux S/AI-2介导的QS系统除了除了参与群体感应外,还与细菌生物发光、成群浮游现象、质粒转移和生物膜的形成、细菌感染过程中与宿主的交叉通讯等。本研究克隆到迟缓爱德华菌luxS基因,长度为516 bp,编码172个氨基酸,预测分子量约19.00kDa,PI为5.993;对基因序列分析结果表明,DNA序列同源性分析显示,luxS基因高度保守,E.t CD的LuxS基因序列与E.t 080813株的同源性为99.8%,与鲶鱼爱德华菌、杀鱼爱德华菌的同源性也都在95%以上。氨基酸序列分析结果显示,E.tarda CD株的LuxS与NCBI上登录的E.tarda 080813株的同源性为100%,与鲶鱼爱德华菌、杀鱼爱德华菌的同源性都在94.8%以上。本研究还进行了原核表达Lux S蛋白,纯化后制备了兔抗LuxS的多克隆抗体,然后用制备的多克隆抗体通过Western blot技术对多株不同来源的迟缓爱德华菌Lux S蛋白的分布情况进行了检测;结果表明,luxS基因在迟缓爱德华菌属中普遍存在。本研究同时通过Western blot技术对不同生长时期迟缓爱德华菌Lux S蛋白表达情况进行了检测,结果表明,LuxS蛋白在不同生长时期的表达并不一致,LuxS蛋白表达量在迟缓期较低,进入对数生长期LuxS蛋白表达量逐渐增加,在对数生长后期LuxS蛋白表达量最大,进入稳定后期Lux S蛋白表达量逐渐减少;此外,本研究还进行了利用外环境添加抗LuxS抗体的方法,探索Lux S是否为信号分子AI-2的特异依赖模式。体外抗体中和生长试验结果表明,1%抗血清(效价1:40000)能延长迟缓爱德华菌生长的平台期,但对细菌生长无显著影响。本研究得出Lux S的关键基因是高度保守的,和luxS/AI-2广泛分布在/迟缓爱德华菌。各生长期luxS基因的表达水平不同,LuxS蛋白表达在指数后期达到最高水平。更多还原

【Abstract】 Edwardsiella tarda is major pathogenic bacteria of aquaculture, after infection, many fish will appear systemic sepsis, the skin ulceration, internal organ necrosis, the whole body severe bleeding; the quorum sensing system(Quorum, sensing, QS) is an important signaling molecule for communication, it is the general language of communication between bacteria,The synthesis mechanism of Autoinducer-2 is only a QS system between gram positive bacteria and gram negative bacteria at the same time; AI-2’s synthetase Lux S is the key of AI-2 synthesis, as well as the inherent component of Activated Methyl Cycle, Lux S/AI-2 system mediated by QS in addition to participate in the induction group, also with the bacterial bioluminescence, plankton, and the phenomenon of plasmid transfer in biofilm formation,infection process and host cross communication and so on.The full-length of AI-2/Lux S of Edwardsiella tarda was cloned by PCR based on the sequence on NCBI, then characteristics and conservative structure of this protein-coding gene were analyzed using web database and bioinformatics tools. The lux S gene was obtained by PCR, its length was 516 bp, and the sequence was highly conversation in Edwardsiella tarda.it encoding a protein of 172 amino acids, with predicted molecular weight of about 19.00 k Da, PI is 5.993; the gene sequence analysis results showed that, lux S gene sequence of E.t CD and E.t 080813 the homology was 99.8%, and Edwardsiella ictaluri and Edwardsiella piscicida homology in more than 95%. NCBI amino acid sequence analysis showed that, the homology between E.t CD strain and E.t 080813 strain were 100%,and Edwardsiella ictaluri and Edwardsiella piscicida homology homology is above 94.8%.This study also conducted a prokaryotic express Lux S proteins, rabbit anti Lux S polyclonal antibody was prepared after purified, then the polyclonal antibody was used in the Western blot technology to detect the Lux S protein from different strains of Edwardsiella tarda, the results show that, the lux S gene was ubiquitous.The anti-rabbits serum was prepared after this protein was purified through prokaryotic expression. The expression level of lux S gene was analyzed during difference growth period using Western blot and the distribution of lux S gene in Edwardsiella tarda was further studiedby this technique. Results of Western blot analysis showed that Lux S expression level was the lowest in the lag phase and began increasing when entered index phase. It reached the peak in the late index phase and decreased in decline phase. To explore whether the specific Lux S is AI-2 dependent, we used the method of antibody neutralization to analyze the effect of antirabbits serum on the growth of Edwardsiella tarda. Moreover, antibody neutralization results showed that, it can elongate the growth plateau phase, but it has no significant effect on bacterial growth.The key gene of lux S was highly conserved, and Lux S/AI-2 was widely distributed among Edwardsiella tarda. The expression level of lux S gene was different during every growth period, expression of Lux S protein reached the highest level in the late index phase.更多还原