【作者】 李杰；

【导师】 莫照兰；

【作者基本信息】 中国科学院研究生院（海洋研究所） ， 水产养殖， 2012， 博士

【摘要】 迟缓爱德华氏菌(Edwardsiella tarda)可引起鱼类的爱德华氏菌病(Edwardsiellosis)，导致出血性败血症。疫苗免疫是预防鱼类病害的重要手段。弱毒疫苗可有效刺激机体系统免疫应答，通过浸泡、口服途径提供有效的免疫保护，实用而高效。以弱毒疫苗为载体可研制鱼类多价疫苗。本研究构建了迟缓爱德华氏菌LSE40的多株基因缺失株，筛选了11株弱毒株并检测它们对鱼的免疫保护效果。初步鉴定了LSE40III型分泌系统(T3SS)效应蛋白EseG的分泌信号肽，为进一步以迟缓爱德华氏菌T3SS为异源抗原呈递途径构建弱毒迟缓爱德华氏菌疫苗载体，进而研制鱼类多价疫苗奠定基础。一、迟缓爱德华氏菌LSE40ΔaroA突变株的特征及免疫效果LSE40ΔaroA突变株缺失了编码5-烯醇丙酮莽草酸-3-磷酸合成酶基因aroA。该突变株的细菌形态变长、分裂延迟，在TSB培养基的生长变慢、运动力和菌膜形成能力减弱，在DMEM培养基中不能自凝、3个T3SS输送器蛋白(EseB，EseC， EseD)的表达和分泌减弱，在鱼体内的存活能力下降，对鱼的毒力减弱。将LSE40ΔaroA以注射和浸泡途径免疫大菱鲆，可检测到免疫鱼的血清产生特异抗体，但免疫鱼不能抵抗致病性迟缓爱德华氏菌的感染。二、迟缓爱德华氏菌LSE40基因缺失株的构建及其毒力检测以迟缓爱德华氏菌LSE40的7个与毒力或营养相关基因(腺苷酸基琥珀酸合成酶编码基因purA，天冬氨酸-β-半醛脱氢酶编码基因asd，5-烯醇丙酮莽草酸-3-磷酸合成酶aroA，T3SS调节蛋白编码基因esrB和装置蛋白编码基因esaC，T6SS装置蛋白编码基因evpH，RNA聚合酶sigma S调节蛋白编码基因rpoS)为靶基因，构建了2株单基因缺失株： LSE40ΔpurA，LSE40Δasd；7株双基因缺失株：LSE40ΔaroAΔesrB、LSE40ΔpurAΔesrB、LSE40ΔpurAΔaroA、LSE40ΔaroAΔesaC、LSE40ΔaroAΔrpoS、LSE40ΔaroAΔevpH、LSE40ΔesrBΔevpH；2株三基因缺失株： LSE40ΔaroAΔesrBΔevpH和LSE40ΔaroAΔesaCΔevpH。以蓝蔓龙检测LSE40ΔaroA和11株突变株的LD50，结果显示毒力下降了130-1000倍以上。三、迟缓爱德华氏菌弱毒株的免疫保护效果首先，以LSE40ΔaroA和11株弱毒株注射免疫蓝蔓龙鱼，发现LSE40ΔaroA、LSE40ΔaroAΔesaCΔevpH和LSE40ΔaroAΔesrBΔevpH三株弱毒株没有提供免疫保护；其他9株弱毒株提供21.7%-82.6%免疫保护率(RPS)。然后，取LSE40ΔaroA和有较高RPS的3株弱毒株LSE40ΔaroAΔesrB、 LSE40ΔaroAΔesaC和LSE40ΔaroAΔevpH浸泡免疫牙鲆，在免疫牙鲆的血清检测到特异性抗体效价，注射攻毒的RPS分别为0、45%、25%和20%，浸泡攻毒的RPS为0、100%、66.7%和66.7%。结果显示，LSE40ΔaroAΔesrB、LSE40ΔaroAΔesaC、LSE40ΔaroAΔevpH对牙鲆提供较好的免疫保护。最后，用LSE40ΔaroAΔesrB注射免疫大菱鲆，108和107cfu/ml免疫组的注射攻毒时免疫保护率分别为20%和10%，106cfu/ml免疫组不能提供免疫保护效果；以浸泡和口服途径免疫大菱鲆，均未能提供免疫保护。综合上述结果，弱毒疫苗对蓝蔓龙和牙鲆提供良好的免疫保护，对大菱鲆未提供免疫保护。四、迟缓爱德华氏菌T3SS效应蛋白分泌性信号肽的鉴定利用细菌的T3SS呈递异源抗原，可构建多价疫苗载体。本实验利用生物信息学方法预测分析LSE40T3SS效应蛋白EseG和输送器蛋白EseC的分泌性信号肽区域，构建待定分泌信号肽与内酰胺酶β-lactamase的融合表达载体，在迟缓爱德华氏菌表达，以westernblot检测融合蛋白的表达和分泌情况。结果显示，EseG的1-88aa具有分泌型信号肽的功能，为进一步研究以迟缓爱德华氏菌T3SS为异源抗原呈递途径打下基础。更多还原

【Abstract】 Edwardsiella tarda can infect farmed fish and develop edwardsiellosis, anenterohaemorrhagic septicaemic disease which result in huge economic losses inaquaculture. Vaccine immunization is an effective approach to prevent E. tardainfection. Attenuated vaccine could stimulate systemic immune response in hostadministrated by the route of immersion or oral, which is use-easy and efficacy in fishaquaculture. Attenuated vaccine is also be used as a vaccine carrier for thedevelopment of the polyvalent vaccine. In this research, I constructed11E. tardamutants by in frame deletion method, and tested their virulence and immuneprotection in fish. For development of attenuated E. tarda as vaccine carrier, weidentified the secretion signal region of EseG, an effector of E. tarda type threesecretion system (T3SS).1Characterization and immune protection of E. tarda LSE40ΔaroA mutantaroA gene encodes5-enolpyruvylshikimate-3-phosphate synthase, which isessential in aromatic amino acid synthesis. We constructed the aroA mutant of E.tarda LSE40and characterized its several phenotypes. Results showed that cellmorphology of the LSE40ΔaroA mutant was elongated and its cell division wasdelayed. This mutant showed lower growth rate in DMEM, less biofilm formation andsmaller swarming diameter. The mutant failed to aggregate as well as to secret threeT3SS translocator proteins (EseB, EseC and EseD) when cultured in DMEM medium.The virulence of LSE40ΔaroA was attenuated in turbot (Scophthalmus maximus) fishaffected by intramuscular injection (i.m.) or immersion. Growth of the mutant wasalso attenuated in turbot. The vaccinated fish administrated by i.m. or immersionshowed high agglutination titres against E. tarda in sera, however, no immuneprotection was observed for both vaccinated groups of fish.2Construction and virulence determination of E. tarda gene deletion mutantsEleven E. tarda LSE40gene deletion mutant were constructed by in framedeletion, including two single-gene-knockout mutants: LSE40ΔpurA and LSE40Δasd, seven double-genes-knockout mutants: LSE40ΔaroAΔesrB, LSE40ΔpurAΔesrB,LSE40ΔpurAΔaroA, LSE40ΔaroAΔesaC, LSE40ΔaroAΔrpoS, LSE40ΔaroAΔevpHand LSE40ΔesrBΔevpH, and two triple-genes-knockout mutants:LSE40ΔaroAΔesrBΔevpH and LSE40ΔaroAΔesaCΔevpH. Gene aroA encodes5-enolpyruvylshikimate-3-phosphate synthase, purA, adenylosuccinate synthase, asd,aspartate-semialdehyde dehydrogenase, rpoS, RNA polymerase sigma factor S, evpH,T6SS apparatus, esrB, T3SS regulator and esaC, T3SS apparatus. Blue gourami(Trichogaster trichopterus) were used to determine the50%lethal dose (LD50) ofthese mutants. Results showed that, these mutants have LD50which was444-1000times higher than the wild type E. tarda LSE40, indicating that all mutants wereattenuated.3Vaccination and immune protection of E. tarda mutantsFirstly, the immune protection of11mutants was evaluated in blue gourami bythe i.m. injection route. Results showed that two mutants, LSE40ΔaroAΔesaCΔevpHand LSE40ΔaroAΔesrBΔevpH did not showed detectable protection for fish; theremaining9mutants could protect blue gourami against wild type E. tarda infection,showing the relative percent of survival (RPS) ranging from21.7%to82.6%. Then,four mutants, LSE40ΔaroA, LSE40ΔaroAΔesrB, LSE40ΔaroAΔesaC, andLSE40ΔaroAΔevpH were evaluated for their immune protection in Japanese flounder(Paralichthys olivaceus) vaccinated by immersion route. Results showed that thespecific antibodies titres against E. tarda could be detected in the sera of vaccinatedfish. The RPS of the four mutants were0,45%,25%and20%against i.m. injectionchallenge of wild-type strain, and0,100%,66.7%and66.7%against immersionchallenge. The results showed that the LSE40ΔaroAΔesrB, LSE40ΔaroAΔesaC andLSE40ΔaroAΔevpH can provide good protection in flounder against wild-type E.tarda LSE40infection. Finally, the immune protection of LSE40ΔaroAΔesrB wasevaluated in turbot vaccinated by the route of immersion or i.m injection or oral.Results showed that immune protection could not be observed in the immersion andoral-vaccinated fish when challenged with wild-type E. tarda. The immune protectioncould be observed in the injection-vaccinated fish with doses of108and107cfu/ml,with RPS in20%and10%, respectively.4Identification the secretion signal of E. tarda T3SS effectorThe attenuated bacterial strains can be used as vaccine vectors for developmentof polyvalent vaccines. T3SS is a potential protein presenting system by delivering the foreign antigens into the host cell. In this study, the secretion signal peptide of T3SSeffector EseG and translocator EseC were predicted by bioinformatics analysis. Thepredicted regions for signal peptide were fused with reporter protein β-lactamase. Thefusion proteins were expressed in E. tarda and their secretion were detected bywesternblot. Result slowed that the N-terminal of1-88amino acid of EseG containedthe secretion signal peptide which could guide the secretion of fusion protein. Next,we will evaluate the potential of E. tarda T3SS as a foreign antigens delivery systemby the EseG signal peptide.更多还原