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《Journal of Fisheries of China》 2012-11
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Construction of Edwardsiella tarda ghosts from eel and their optimization of preparation conditions

LI Ning-qiu,YU Lu-jun,FU Xiao-zhe,LIU Li-hui,LIN Qiang,CHANG Ou-qin,SHI Cun-bin,HUANG Zhi-bin,WU Shu-qin(Key Laboratory of Fishery Drug Development,Ministry of Agriculture,Key Laboratory of Aquatic Animal Immune Technology,Guangdong Province,Pearl River Fisheries Research Institute,Chinese Academy of Fishery Sciences,Guangzhou 510380,China)  
Edwardsiellosis caused by Edwardsiella tarda is one of the most serious diseases in the eel culture 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 transformants of E.coli and E.tarda carrying plasmid pBVlysisE was performed successfully by increasing the incubation temperature up to 42℃.Compared to E.coli,in which lysis was observed within 30 min and was completed 3 h after induction of E gene expression,onset of E.tarda lysis occurred 1 h after temperature 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 experiments 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.
【Fund】: 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)
【CateGory Index】: S948
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