基于AhsA蛋白的嗜水气单胞菌特异性抗体的制备与鉴定

选取嗜水气单胞菌表面特异性抗原蛋白AhsA,构建了pET-28a(+)-AhsA表达载体,并在大肠杆菌BL21中进行表达,利用Ni-NTA亲和层析、凝胶过滤层析等蛋白纯化方法进行纯化。重组蛋白表达效果较好,浓度可达10 mg·mL^-1,纯度可达90%以上,符合制备多克隆抗体的免疫原标准。利用Western Blot技术将AhsA抗体特定识别免疫原蛋白,结果表明该抗体能够特异性识别免疫原蛋白AhsA,而对于牛血清蛋白却不能发生免疫应答,表明该抗体具有一定的特异性且AhsA蛋白具有很好的免疫原性。同时通过检测验证了AhsA蛋白抗体能够特异性检测嗜水气单胞菌,将为水产品质量控制及临床检测嗜水气单胞菌可提供新的技术。

Preparation and Identification of AhsA Polyclonal Antibodies

In this paper, in order to obtain the surface specific antigen protein AhsA of Aeromonas hydrophila, pET-28a (+) -AhsA expression vector was constructed and was expressed in Escherichia coli BL21, then AhsA purified by Ni⁺ affinity chromatography and gel filtration chromatography. The recombinant protein had a good expression effect with a concentration of 10 mg·mL^-1 and a purity of more than 90%, meeting the immunogen standard for preparation of polyclonal antibodies. Western Blot assay was used to identify AhsA antibody to specific immunogenic protein, and the results showed that this antibody could specifically identify AhsA, but could not produce an immune response to bovine serum protein, indicating that this antibody had certain specificity and AhsA protein had good immunogenicity. The other results revealed that the antibody only recognize A. hydrophila. AhsA antibody will provide a new technique for quality control of aquatic products and clinical detection of A. hydrophila.