Mao Zhijuan, Yu Lian, You Zhenqiang, Wei Yongwei, Liu Yan
College of Biomedical Engineering and Instrument Science, Zhejiang University, Hangzhou, Zhejiang Province 310027, China.
Fish Shellfish Immunol. 2007 Sep;23(3):567-75. doi: 10.1016/j.fsi.2007.01.004. Epub 2007 Jan 20.
Genes of five outer membrane proteins of Vibrio parahaemolyticus zj2003, including OmpW, OmpV, OmpK, OmpU and TolC, were cloned and expressed as N-terminal His(6)-tagged proteins in Escherichia coli. The recombinant fusion proteins were purified with nickel chelate affinity chromatography. To analyze the immunogenicity of these proteins, large yellow croaker (Pseudosciaena crocea) were immunized by intraperitoneal injection. Antibody response was assessed by method of enzyme-linked immunosorbent assay. Titres to all five recombinant proteins increased during 4 to 8 weeks post immunization, within the range of log 2 values of 5.75 to 10.8. Recorded relative survival percent (RPS) of the vaccinated groups varied from 80% to 90%, while 10 fish in control group all died. Western blot tests were undertaken with the serum of survival fish after experimental infection. Except for recombinant TolC, the other four recombinant proteins were recognized by the serum. It is indicated that four outer membrane proteins of V. parahaemolyticus zj2003, including OmpW, OmpV, OmpU and OmpK, are immunogenic during in vivo infection, which would be of some significance in developing efficient vaccine in aquaculture. This is the first report of successful vaccination against V. parahaemolyticus with purified recombinant outer membrane proteins.
对副溶血性弧菌zj2003的5种外膜蛋白基因，包括OmpW、OmpV、OmpK、OmpU和TolC进行了克隆，并在大肠杆菌中表达为N端带有His(6)标签的蛋白。重组融合蛋白通过镍螯合亲和层析进行纯化。为分析这些蛋白的免疫原性，通过腹腔注射对大黄鱼（Pseudosciaena crocea）进行免疫。采用酶联免疫吸附测定法评估抗体反应。免疫后4至8周内，所有5种重组蛋白的抗体效价均有所升高，log2值范围为5.75至10.8。接种组的相对存活百分比（RPS）记录值在80%至90%之间，而对照组的10条鱼全部死亡。对实验感染后存活鱼的血清进行了蛋白质免疫印迹试验。除重组TolC外，其他4种重组蛋白均能被血清识别。结果表明，副溶血性弧菌zj2003的4种外膜蛋白，包括OmpW、OmpV、OmpU和OmpK，在体内感染过程中具有免疫原性，这对于水产养殖中开发高效疫苗具有一定意义。这是首次报道用纯化的重组外膜蛋白成功接种预防副溶血性弧菌的研究。
