[Objective] As one of the key enzymes of the glycolysis in E.tarda,GAPDH was previously identified to be a broad-spectrum antigen and could be used as the target of bacterial vaccine design in aquaculture.We explore the mechanism of secretion of GAPDH in E.tarda.[Methods] Secretion of GAPDH in E.tarda EIB202 and mutants of several secretory pathways were analyzed by Western blot and ELISA.The levels of GAPDH in the supernatants of mutant library were analyzed through high-throughput screening.Quantitative real-time PCR was used to compare the mRNA expressions of mutants obtained from screening.[Results] Secretion of GAPDH in E.tarda is associated with classical secretion systems.Through high-throughput screening,two mutants △esrA and △esrC were found to have higher levels of GAPDH in the supernatants.Lacking of these two genes lead to significant up-regulation of secretion of GAPDH.[Conclusion] EsrA and EsrC both played a negative regulation role in GAPDH secretion.%[目的]迟缓爱德华氏菌甘油醛-3-磷酸脱氢酶(GAPDH)是糖酵解途径中关键酶之一,前期研究证实是一种广谱性抗原,可作为水产养殖细菌病免疫防治中疫苗的开发靶点.本文探究迟缓爱德华氏菌甘油醛-3-磷酸脱氢酶的胞外分泌机制.[方法]通过Western blot和ELISA方法考察迟缓爱德华氏菌经典分泌系统缺失株GAPDH胞外分泌情况;使用ELISA方法对迟缓爱德华民菌突变体文库的GAPDH胞外分泌进行了大规模筛查,并结合qRT-PCR对筛查得到的插入失活株进行了表达分析.[结果]经典分泌系统与GAPDH的胞外分泌存在一定相关性.突变体文库的大规模筛查得到两株GAPDH分泌量明显增加的插入失活株AesrA和△esrC,这两个基因的失活会导致GAPDH的胞外分泌量显著上调.[结论]迟缓爱德华民菌GAPDH的胞外分泌受EsrA和EsrC负调控.
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