Molecular characterization of the Aeromonas hydrophila aroA gene and potential use of an auxotrophic aroA mutant as a live attenuated vaccine
Molecular characterization of the Aeromonas hydrophila aroA gene and potential use of an auxotrophic aroA mutant as a live attenuated vaccine
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DOI：
10.1128/iai.66.5.1813-1821.1998
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发表时间：
1998-05-01
影响因子：
3.1
通讯作者：
Carrasco, GN
中科院分区：
文献类型：
--
作者：
Moral, CH;Del Castillo, EF;Carrasco, GN
利用大肠杆菌K-12菌株AB2829的aroA突变，克隆了嗜水气单胞菌SO2/2编码5-烯醇丙酮酰石草酸3-磷酸合酶的aroA基因，并测定了其核苷酸序列。该基因编码一个440个氨基酸的蛋白，与其他细菌aroA蛋白具有高度的同源性。通过插入含有卡那霉素抗性决定因素的DNA片段灭活aroA基因，并通过等位基因交换，通过自杀载体pSUP202将aroA基因重新引入嗜水单胞菌AG2的染色体中，获得了一种有效的抗鱼嗜水单胞菌感染的减毒活疫苗。嗜水芽胞杆菌突变体AG2 aroA::Ka(r)以50%致死剂量bbb20 × 10(8) CFU腹腔接种虹鳟鱼后，具有高度减毒作用。接种后48 h，突变体不能从内脏中恢复。免疫组织化学研究表明，在注射后9天，肾脏和脾脏中存在免疫阳性物质，而不是整个细胞，与一种针对嗜水单胞杆菌的多克隆抗血清反应。用AroA突变体接种虹鳟鱼作为活疫苗，对野生型嗜水芽胞杆菌具有显著的保护作用。
The aroA gene of Aeromonas hydrophila SO2/2, encoding 5-enolpyruvylshikimate 3-phosphate synthase, was cloned by complementation of the aroA mutation in Escherichia coli K-12 strain AB2829, and the nucleotide sequence was determined. The nucleotide sequence of the A. hydrophila aroA gene encoded a protein of 440 amino acids which showed a high degree of homology to other bacterial AroA proteins. To obtain an effective attenuated live vaccine against A. hydrophila infections in fish, the aroA gene was inactivated by the insertion of a DNA fragment containing a kanamycin resistance determinant and reintroduced by allelic exchange into the chromosome of A. hydrophila AG2 by means of the suicide vector pSUP202. The A. hydrophila mutant AG2 aroA::Ka(r) was highly attenuated when inoculated intraperitoneally into a rainbow trout, with a 50% lethal dose of >2 x 10(8) CFU. The mutants were not recoverable from the internal organs after 48 h postinoculation. Immunohistochemical studies demonstrated that immunopositive materials, but not whole cells, reacting with a polyclonal antiserum against A. hydrophila were present in the kidney and spleen 9 days postinjection. Vaccination of rainbow trout with the AroA mutant as a live vaccine conferred significant protection against the wild-type strain of A. hydrophila.