鱼源嗜水气单胞菌aopB-/aopD-/aroA-缺失株的构建及鉴定

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。

Construction and Characterization of aopB-/aopD-/aroA-Deleted Mutant Strain in Aeromonas hydrophila

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 1290 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.