Cloning, expression and immunogenicty analysis of five outer membrane proteins of Vibrio parahaemolyticus zj2003.
Cloning, expression and immunogenicty analysis of five outer membrane proteins of Vibrio parahaemolyticus zj2003.
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DOI：
10.1016/j.fsi.2007.01.004
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发表时间：
2007-09
影响因子：
4.7
通讯作者：
Zhijuan Mao;Lian Yu;Z. You;Yongwei Wei;Yan Liu
中科院分区：
文献类型：
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作者：
Zhijuan Mao;Lian Yu;Z. You;Yongwei Wei;Yan Liu
克隆了副溶血性弧菌（Vibrio parahaemolyticus）zj 2003的5种外膜蛋白OmpW、OmpV、OmpK、OmpU和TolC的基因，并在大肠杆菌中进行了表达。重组融合蛋白经镍螯合亲和层析纯化。以大黄鱼（Pseudosciaena crocea）为材料，通过腹腔注射免疫，分析其免疫原性。采用酶联免疫吸附试验（ELISA）检测抗体反应。所有五种重组蛋白的滴度在免疫后4至8周期间增加，在log 2值5.75至10.8的范围内。接种组的相对存活率（RPS）为80%-90%，而对照组中的10条鱼全部死亡。用实验感染后存活鱼的血清进行Western印迹试验。除重组TolC外，其余4种重组蛋白均能被血清识别。结果表明，副溶血性弧菌zj 2003的4种外膜蛋白OmpW、OmpV、OmpU和OmpK在体内感染时具有免疫原性，这对研制高效疫苗具有一定的意义。这是第一次报告成功的疫苗接种副溶血性弧菌与纯化的重组外膜蛋白。
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 8weeks post immunization, within the range of log2 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.