嗜水气单胞菌BYKAH2008AC株外膜蛋白和溶血素双基因的融合表达及免疫原性分析

Fusion expression and immunogenicity analysis of OmpTS gene with Hly gene of Aeromonas hydrophila strain BYKAH2008AC

嗜水气单胞菌(Aeromonas hydrophila)的外膜蛋白基因(OmpTS)和溶血素基因(Hly)是其重要的保护性抗原。根据Genbank已发表的两个基因的序列设计引物,扩增其去除信号肽部分的基因片段,再将二者通过柔性片段融合,定向插入到质粒pET32a中构建重组融合表达载体,并转化到大肠杆菌BL21(DE3),经IPTG诱导获得与预期大小108 kD相吻合的融合蛋白条带。用纯化的重组蛋白免疫新西兰大白兔制备兔抗血清。ELISA和Western Blot检测该抗体效价为1∶4000以上,且该重组蛋白与抗OmpTS兔血清和抗Hly兔血清都呈阳性反应,表明该融合蛋白保留了外膜蛋白和溶血素的免疫原性,可作为基因工程亚单位疫苗的候选成分。

Outer membrane protein and hemolysin was proven to be protective antigens against Aeromonas hydrophila. Primers were designed according to the gene sequence of OmpTS and Hly of A. hydrophila published in GenBank. With the specific primers, their coding regions were amplified by PCR method and cloned in to pET32a vector linking by flexible clips. After transformed into E. coli DH5a and induced with IPTG, the 108Kd fusion engineered protein was expressed as expected. After purified by Ni^2＋ affinity chroma- tography, the protein was used to immunize the rabbit to produce antiserum. The ELISA result showed that the titer of antiserum was above 1：4000. Western blot analysis showed that rabbit antiserum to OmpTS and Hly all reacted with this fusion engineered protein, which indicated the fusion protein may have similar epitopes to these natural proteins. All these findings suggested that this recombinant OmpTS-Hly had the potential to be candidate components of an effective vaccine in fish against the infection of A. hydrophila.