鳗源迟缓爱德华氏菌菌蜕的构建及制备条件优化  

Construction of Edwardsiella tarda ghosts from eel and their optimization of preparation conditions

作　　者：李宁求[1] 余露军[1] 付小哲[1] 刘礼辉[1] 林强[1] 常藕琴[1] 石存斌[1] 黄志斌[1] 吴淑勤[1] 

机构地区：[1]中国水产科学研究院珠江水产研究所,农业部渔药创制重点实验室,广东省免疫技术重点实验室,广东广州510380

出　　处：《水产学报》2012年第11期1754-1762,共9页Journal of Fisheries of China

基　　金：国家科技支撑计划项目(2012BAD25B02);国家自然科学基金项目(31202032);广东省自然科学基金项目(7004728)

摘　　要：菌蜕具有完整细菌表面抗原结构,可诱导机体的体液和细胞免疫应答,成为疫苗制备的候选之一,为了探讨鳗鲡迟缓爱德华氏菌菌蜕疫苗的可行性,实验采用基因重组技术构建了噬菌体PhiX174裂解酶基因(Lysis E)的温控表达载体,转化迟缓爱德华氏菌,成功制备其菌蜕,并对菌蜕形态、溶菌动力学、裂解效率以及制备条件等进行了研究。结果表明,细菌菌蜕表面形成溶菌孔道,细胞因内容物流失而发生明显的皱缩;构建的迟缓爱德华氏菌诱导后1 h开始裂解,5 h后裂解基本完成,裂解效率为99.99%,冷冻干燥后重悬涂布平板,未检出活菌,电镜观察表明冻干前后细胞形态未见明显变化;构建的迟缓爱德华氏菌分别在OD600值为0.4和0.6进行诱导,其裂解过程和裂解效率没有明显区别;分别用LB、BHI、NB 3种培养基进行比较研究,其中LB培养基制备的菌蜕细胞较完整、裂解完全,是制备迟缓爱德华氏菌菌蜕最优培养基。本研究成功构建了鳗源迟缓爱德华氏菌菌蜕,并对其制备条件进行了优化,为鳗鲡爱德华氏菌病疫苗开发奠定了基础。Edwardsiellosis caused by Edwardsiella tarda is one of the most serious diseases in the eel cul- ture industry. Bacterial ghosts, which have native surface antigenic structures and can induce humoral and cellular immune response, have become candidate of vaccine development. To explore the feasibility of E. tarda ghost vaccine in eel, E. tarda ghosts were generated by the thermal controlled expression of the PhiX174 lysis gene E in present study. At the same time the morphology, lysis kinetics, lysis efficiency and preparation conditions were also studied. Holes were observed in Escherichia coli and E. tarda ghosts by scanning electron microscopy and the bacteria become withered due to the loss of bacterial content. The lysis kinetics in E. tarda were also compared with those in E.coli. Generation of ghosts in the transfor- mants of E. coli and E. tarda carrying plasmid pBVlysisE was performed successfully by increasing the incubation temperature up to 42~C. Compared to E. coli, in which lysis was observed within 30 min and was completed 3 h after induction orE gene expression, onset orE. tarda lysis occurred 1 h after tempera- ture elevation and the lysis process was completed 5 h after induction. At the end of the lysis process, the efficiency of ghost induction in non-lyophilized E. tarda was 99.99%, as the results of 5 replicate experi- ments showed. However, no bacterial growth was detected in lyophilized E. tarda and there is no obvious difference in bacterial morphology before and after lyophilization. The lysis kinetics and lysis efficiency had no difference between beginning of induction at OD600=0.4 and 0.6. Among LB, BHI and NB medium, LB medium is the best for preparation of E. tarda because in this medium the ghosts are more integrated and lysed completely. In the study we successfully constructed E. tarda ghosts and optimized their preparation conditions, which will provide the foundation for development of vaccine against edwardsiellosis in eel.

关 键 词：鳗鲡 迟缓爱德华氏菌 菌蜕 条件优化 

分 类 号：Q784[生物学—分子生物学] S917.4[农业科学—水产科学]