溶藻弧菌减毒活疫苗的构建及其免疫保护性

Construction of live attenuated Vibrio alginolyticus vaccine and its immunogenic and protective effects

采用Overlap PCR和同源重组技术,结合氯霉素(Cm)正向筛选和sacB基因蔗糖负向筛选,构建了溶藻弧菌(Vibrio alginolyticus)Ⅲ型分泌系统(Type III secretion system,T3SS)注射装置蛋白vscO基因无标记框内缺失突变株ZJ03ΔvscO。在以石斑鱼(Epinephelus coioides)为模型的感染实验中发现ZJ03ΔvscO的毒力较ZJ03下降约150倍。将ZJ03ΔvscO作为减毒活疫苗通过注射、浸泡的途径免疫石斑鱼(Epinephelus coioides)后,发现其对溶藻弧菌有很好的保护效应,其中注射组的免疫保护率达84%,浸泡组次之(68%)。血清效价分析显示,免疫后第4周注射组和浸泡组的效价均达到最高值,分别为1:2 048和1︰128。上述结果表明,所构建的ZJ03ΔvscO减毒活疫苗可有效提高石斑鱼对溶藻弧菌的免疫力,并且免疫和攻毒的途径可能是影响免疫保护效果的因子。本研究旨在为溶藻弧菌引起的鱼类疾病防治提供技术支撑。

Vibrio alginolyticus, a gram-negative bacterium has been described to be among the most common and eco-nomically important aquatic pathogen of fish and shellfish. Vaccine immunization is an effective approach to prevent V. alginolyticus infection. And attenuated vaccine could stimulate systemic immune response in host administrated. Now there has been little attenuated vaccine of V. alginolyticus. The type III secretion system （T3SS） is used by bacterial pathogens to inject effector proteins into the cytoplasm of their host cells. And it is an important pathogenic factor of Vibrio alginolyticus. The T3SS consists of a structure called needle complex, which is made of a multi-ring base that spans the bacterial and a needle-like extension that protrudes several nanometers from the bacterial surface. VscO is located at the“needle”site of T3SS. Its role is to escort the molecular chaperone and effector proteins into host cells, and further induced host cell death. In this paper, combining with chloramphenicol （Cm） and sucrose screening, an in-frame deletion mutant of vscO using overlap PCR and homologous recombination technology was constructed. And the LD50 changes of ZJ03ΔvscO mutant strains compared with the strain ZJ03 were examed. Grouper （Epinephelus coioides）, body length （17±3） cm, body weight （50±5） g were injected with ZJ03ΔvscO and ZJ03, respectively. TheΔvscO mutant showed about 150 times decrease in virulence in grouper. The ZJ03ΔvscO was used to immunize grou-per by way of injection and immersion. The result indicated that the specific antibody titers were markedly higher than that in the control group （P〈0.05）.The relative percentage survival （RPS） of injection group was 84%, while the RPS of immersion group was 68%. The results showed that the ZJ03ΔvscO of V. alginolyticus has a high immunogenicity, and can be a live attenuated vaccine. In addition, immunity and infection way may be the factor affecting the RPS. The purpose of this study is to provide technical support for fish diseases caused by V. alginolyticus .