【摘要】 嗜水气单胞菌(Aeromonas hydrophila)能引起多种鱼类暴发性出血性败血症,为研制更加安全的嗜水气单胞菌口服活疫苗,构建了鱼源嗜水气单胞菌aopB-/aopD-/aroA-缺失株。首先构建含缺失369 bp的aopB/aopD基因(编码嗜水气单胞菌外膜蛋白B、D)与蔗糖敏感基因(sacB)的重组自杀性质粒pREaopB/aopD,与嗜水气单胞菌Ah2056接合转移,应用二步法筛选aopB-/aopD-缺失株,PCR证实aopB/aopD基因的缺失突变。在此基础上,构建另一个含缺失1 290 bp的aroA基因(编码5-酸烯醇式丙酮酸莽草酸-3-磷酸合酶)与庆大霉素抗性片段(aacC1片段)的重组自杀性质粒pSUParoA,与aopB-/aopD-缺失株接合转移,利用庆大霉素抗性筛选aopB-/aopD-/aroA-缺失株,用PCR证实aroA基因的缺失突变。进一步对aopB-/aopD--/aroA-缺失株进行生长特性、对鱼的毒力等生物学特性鉴定。结果表明,aopB-/aopD-/aroA-缺失株构建成功,且该缺失株为毒力丧失、营养缺陷株。更多还原

【Abstract】 Aeromonas hydrophila is a gram-negative opportunistic pathogen that can result in motile aeromonad septicemia in numerous freshwater fish species.In order to develop a safer vaccine strain for oral immunization,aopB-,aopD-and aroA-triple-deletion mutant was constructed.Firstly,the recombination suicide vector pREaopB/aopD with sacB gene(sucrose-sensitive) and 369 bp-deleted aopB-/aopD-gene(encoding A.hydrophila out membrane protein B,D) was constructed and conjugated with Ah2056.The aopB/aopD deleted strain was selected by two-step method and identified by PCR.Then,the other recombination suicide vector pSUParoA with gentamycin resistance gene and 1 290 bp-deleted aroA gene(encoding 5-enolpyruvyl shikimate 3-phosphate synthase) was constructed and conjugated with the aopB-/aopD-deleted strain.The aopB-/aopD-/aroA-deleted strain was selected by gentamycin and identified by PCR.The growth properties and virulence in fish of the aopB-/aopD-/aroA-deleted strain were further characterized.Results showed that the aopB-/aopD-/aroA-deleted mutant was successful constructed.The mutant was lower virulence and aromatic amino acids dependent.The mutant can be potentially used as an oral vaccine strain.更多还原