嗜水气单胞菌J-1株OmpA融合蛋白的高效表达及免疫原性分析

Expression and immunogenicity analysis of the fusion protein OmpA from Aeromonas hydrophila strain J-1

以嗜水气单胞菌（Aeromonas hydrophila）J-1株基因组为模板，根据GenBank已发表的嗜水气单胞菌外膜蛋白的基因序列设计1对引物，扩增去除信号肽序列的成熟外膜蛋白OmpA的基因片段，定向克隆至表达质粒pET32a（＋）中，重组质粒经限制性酶切鉴定后测序。结果表明，插入片段长度为948bp，与NCBI上登录的几个相关序列相比，同源性在93．1％-95％之间，缺失4个氨基酸。将重组原核表达质粒pET32a-OmpA转化至大肠杆菌BL21（DE3），经诱导可表达分子量约57．4kD的蛋白，凝胶薄层扫描显示OmpA在转化菌中表达量占全菌蛋白的50％以上。用兔抗J-1株总外膜蛋白血清进行Western blot，在57．4kD处有明显反应条带。融合蛋白经Ni-NTA树脂柱亲和层析纯化后免疫新西兰兔制备抗血清，ELISA效价达1：12800以上。Western blot检测结果显示，该血清与9株具有代表性的气单胞菌的分子量约35kD的外膜蛋白均有较强反应，说明所表达的融合蛋白不仅保持原有外膜蛋白的免疫原性，而且提示OmpA可能是嗜水气单胞菌的共同保护性抗原。[中国水产科学，2008，15（2）：301—306]

Aeromonas hydrophila is a significantly important pathogen for freshwater fish and human. Outer membrane proteins from Aeromonas spp. have been identified as suitable candidate for vaccine development in fish.In ordor to express the outer memberane protein A （OmpA） of A.hydrophila strain J-1 and examine its immunogenicity, primers were designed according to the gene sequence of OmpA of A.hydrophila published in GenBank. With the specific primers, the coding region of OmpA gene from A. hydrophila strain J-1 was amplified by PCR method and cloned into pET-32a （＋） vector. The recombinant plasmid pET32-OmpA was transformed into E.coli BL21 （DE3） and induced with IPTG. The sequence analysis revealed that the cloned gene had 93.1%-95.0% homology with the sequences of OMPs from other strains ofA. hydrophila, and lacked 4 amino acid residues in two sites. SDS-PAGE and western blot analysis showed that the target recombinant protein with the molecular weight of 57.4 kD was hyperexpressed in the form of inclusion body. After purified by Ni^2＋-affinity chromatography, the protein was used to immunize the rabbit. Antiserum was collected and used to detect the specificity of recombinant protein. ELISA result showed that the titer of antiserum was above 1 ： 12 800. Moreover, western blot result reveded that the antiserum reacted not only specifically with the purified recombinant protein but also with about 35 kD OMPs from 8 strains ofA. hydrophila, involving 3 major serotypes in China and one strain of A.salmonicida. The result indicates that recombinant OmpA has the same immunity epitopes as natural one and may be the common protective antigen in different Aeromonas isolates. [Journal of Fishery Sciences of China, 2008, 15 （2） ： 301-306]