副溶血弧菌融合蛋白FlaA-OmpK疫苗的制备及免疫保护作用  

Expression and immune characteristics of FlaA-OmpK protein in Vibrio parahaemolyticus

作　　者：郑磊[1,2] 郭养浩[1] 马振宁[1] 樊海平[2] 吴斌[2] 唐凤翔[1] 

机构地区：[1]福州大学药物生物技术与工程研究所,福建福州350002 [2]福建省淡水水产研究所,福建福州350002

出　　处：《中国水产科学》2012年第5期848-853,共6页Journal of Fishery Sciences of China

基　　金：福建省科技厅计划重点项目(2010N0014);福建省教育厅A类科技项目(JA10024)

摘　　要：采用延伸PCR技术拼接副溶血弧菌(Vibrio parahaemolyticus VpATCC17802)的外膜蛋白K基因ompK和鞭毛蛋白A基因flaA,获得融合基因flaA-ompK。制备高纯度的r-OmpK,r-FlaA及r-FlaA-OmpK蛋白。分别以所制备的OmpK、FlaA-OmpK和混合蛋白OmpK+FlaA作为免疫原,通过口服及注射的方法免疫黑石斑鱼(Centropristisstriata),研究其免疫原性及对野生副溶血弧菌(Vp89)感染的免疫保护作用。ELISA分析结果表明,注射FlaA-OmpK组抗体效价最高,是OmpK组的2倍,是混合蛋白组的4倍,注射FlaA-Ompk提供的免疫保护率达到80%。本工作制备了FlaA-Ompk肠溶口服微球疫苗,口服免疫组血清抗体效价低于注射组血清抗体效价,口服FlaA-Ompk提供的免疫保护率达到50%。研究结果显示融合蛋白FlaA-Ompk具有良好的免疫原性,可作为多元弧菌疫苗抗原成份。Marine vibrio is a causative agent of vibriosis, a disease that results in severe losses to the aquaculture industry. The development of Vibrio vaccines would likely lower the abuse of antibiotics that are currently used to ensure the safety of aquatic products. We prepared and characterized a FlaA-OmpK enteric microsphere vaccine and evaluated the effectiveness of oral vaccination. We cloned the ompK andflaA genes from the total DNA of V. parahaemolyticus. FlaA was fused with ompK intoflaA-ompK by overlap extension PCR. The expression systems OmpK, FlaA, and FlaA-OmpK were then constructed. The recombinant proteins were expressed in large scale in E coli BL21 and purified. OmpK, FlaA-OmpK, and mixed OmpK and FlaA were administered as antigens to immu- nize Centropristis striata. Vaccination with FlaA-OmpK protected against the infection by V. parahaemolyticus. The titers of the anti-serum from immunized C. striata against FlaA-OmpK were highest 40 d after immunization, and were two times that of the OmpK group and four times that of the OmpK group. The serum titers of the FlaA-Ompk were significantly lower in fish that were treated orally relative to those that were injected. The fish immunized with r-FlaA-OmpK had high survival （80%） compared with the control group. Similarly, the oral FlaA-OmpK protection rate （50%） was lower than that in the injection group. Our results suggest that If. para- haemolyticus FlaA-Ompk is an ideal candidate for development of a Vibriosis vaccine.

关 键 词：副溶血弧菌 融合蛋白FlaA-Ompk 免疫原性 口服疫苗 

分 类 号：S942[农业科学—水产养殖]