鱼源嗜水气单胞菌aopB-／aopD-／aroA-缺失株的构建及鉴定  

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

作　　者：罗晓松[1,2] 尚书[1] 侯化鹏[1] 汪登强[2] 邹世平[1,2] 曾令兵[2] 龙良启[1] 

机构地区：[1]华中农业大学动物医学院,湖北武汉430070 [2]中国水产科学研究院长江水产研究所,荆州434000

出　　处：《农业生物技术学报》2009年第1期29-35,共7页Journal of Agricultural Biotechnology

基　　金：农业部结构调整重大技术研究专项(No.:06-05-03B);中国水产科学研究院淡水生态重点实验室基金(No.2006-A-12)资助

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。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.

关 键 词：嗜水气单胞菌 自杀性质粒pRE112 自杀性质粒pSUP202 aopB-/aopD-/aroA-缺失株 

分 类 号：S188[农业科学—农业基础科学]