副溶血弧菌(Vibrio parahaemolyticus)tdh2基因和鳗弧菌(V.anguillarum)ompU基因二联DNA疫苗制备及其对大菱鲆(Scophthalmus maximus)免疫保护作用

CONSTRUCTION AND PROTECTION EVALUATION OF A BIVALENT DNA VACCINE CONTAINING tdh2 OF VIBRIO PARAHAEMOLYTICUS AND ompU OF V.ANGUILLARUM ON TURBOT SCOPHTHALMUS MAXIMUS

采用基因工程的方法,将副溶血弧菌的热稳定直接溶血素基因tdh2和鳗弧菌外膜蛋白基因ompU进行融合,在大肠杆菌中得到表达,并利用该融合基因构建二联DNA疫苗pEGFP-N1/tdh2-ompU。用DNA疫苗按10和50μg/尾的剂量通过肌肉注射免疫大菱鲆,对大菱鲆抵抗致病性副溶血弧菌和鳗弧菌的免疫效果进行研究。结果表明,DNA疫苗免疫的大菱鲆对副溶血弧菌感染的最高保护率为100%,对鳗弧菌感染的保护率为35%。被免疫的大菱鲆肌肉组织中能检测到融合蛋白表达,在血清中能检测到较高水平特异性抗体,DNA疫苗引起的体液免疫反应水平和保护效果与注射剂量有关。

Vibriosis is one of the most prevalent fish diseases caused by bacteria that belong to the genus Vibrio, in which Vibrio parahaemolyticus and E anguillarum are two most common pathogenic agents. In this study, a thermostable direct hemolysin （TDH） gene tdh2 of V. parahaemolyticus and an outer membrane protein （OMP） gene ompU of V. anguillarum were inserted into prokaryotic expression vector pET28a （＋） successively. Then the gene encoding the fusion protein TDH2-OmpU was used to construct bivalent DNA vaccine. The protective efficiency of such vaccine against V. parahaemolyticus and V. anguillarum was evaluated in turbot Scophthalmus maximus. Fish immunized with a single intramuscular injection of different doses of the tdh2-ompU DNA vaccine showed significantly higher serum antibody levels after vaccination, compared to fish injected with the control eukaryotic expression vector pEGFP-N1. Four weeks after inoculation, turbot vaccinated with the bivalent DNA vaccine were exposed to V. parahaemolyticus and V. anguillarum by intraperitoneal injection and intramuscular injection, respectively. The highest relative percent survival （RPS） rates, 100% against V. parahaemolyticus and 35% against V. anguillarum, respectively, were recorded. Furthermore, humoral immune responses and protective effects significantly increased at higher vaccine doses.