Zhang Chongwen, Yu Lian, Qian Ronghua
College of Biomedical Engineering and Instrument Science and Institute of Preventive Veterinary Medicine, Zhejiang University, Hangzhou 310027, China.
J Aquat Anim Health. 2008 Mar;20(1):1-11. doi: 10.1577/H06-031.1.
Vibrio harveyi is a causative agent of vibriosis in the large yellow croaker Pseudosciaena crocea and causes severe losses to the aquaculture industry in China. The vaccines based on the outer membrane proteins (OMPs) of the pathogens are considered to be the optimum intervention for this disease. In this study, two V. harveyi OMP genes, OmpK* and glyceraldehyde-3-phosphate dehydrogenase (GAPDH*), were cloned, sequenced, and characterized. The recombinant proteins (r-OmpK and r-GAPDH) were expressed by the prokaryotic expression vector pET-30a(+) and purified with nickel-nitrilotriacetic acid affinity chromatography. Western blots showed that rabbit antisera against purified r-OmpK and r-GAPDH specifically reacted with the native OMP of V. harveyi. Large yellow croakers were immunized with r-OmpK and r-GAPDH. Specific antibody titer assessed by enzyme-linked immunosorbent and phagocytosis assays demonstrated that specific and innate immunity was stimulated in response to the OMPs of V. harveyi. Challenge results indicated that vaccination of large yellow croakers with r-OmpK and r-GAPDH increased relative survival (37.7% and 40.0%, respectively) against wild V. harveyi.
哈维氏弧菌是大黄鱼弧菌病的病原体，给中国水产养殖业造成了严重损失。基于病原体外膜蛋白（OMPs）的疫苗被认为是针对这种疾病的最佳干预措施。在本研究中，克隆、测序并鉴定了两个哈维氏弧菌OMP基因，即OmpK和甘油醛-3-磷酸脱氢酶（GAPDH）。重组蛋白（r-OmpK和r-GAPDH）通过原核表达载体pET-30a(+)表达，并用镍-亚氨基三乙酸亲和层析法纯化。蛋白质免疫印迹显示，针对纯化的r-OmpK和r-GAPDH的兔抗血清与哈维氏弧菌的天然OMP发生特异性反应。用r-OmpK和r-GAPDH对大黄鱼进行免疫。通过酶联免疫吸附和吞噬试验评估的特异性抗体效价表明，针对哈维氏弧菌OMPs可刺激特异性免疫和固有免疫。攻毒结果表明，用r-OmpK和r-GAPDH对大黄鱼进行疫苗接种可提高其对野生哈维氏弧菌的相对存活率（分别为37.7%和40.0%）。
