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迟缓爱德华氏菌是水产养殖中危害性极大的一种致病菌,该菌的两种外膜蛋白GAPDH和ETA1被证明可以独立产生免疫原性,具有开发成疫苗的潜力。文章将这两个蛋白融合表达以期增强疫苗的保护作用。采用PCR方法扩增出融合有gapdh基因和eta1基因的融合基因rh,将该目的基因与表达载体p ET-30a(+)连接,转入大肠杆菌BL21(DE3)菌株中表达融合蛋白,采用SDS-PAGE检测融合蛋白的表达,并用镍柱和葡聚糖凝胶Sephadex G-100对融合蛋白进行了纯化,最后通过Western-blot对纯化的融合蛋白进行了验证。结果表明融合蛋白主要以包涵体形式表达,在破碎后的菌体沉淀中该蛋白含量高达73.6%,经过纯化后的融合蛋白纯度高达90%,并通过Western-blot得到了验证。上述结果表明表达载体构建成功,目的蛋白在大肠杆菌中大量表达。
Agrobacterium tumefaciens is a highly virulent pathogen in aquaculture. The two outer membrane proteins, GAPDH and ETA1, proved to be independently immunogenic and have the potential to be developed into vaccines. The article expressed these two proteins in order to enhance the protection of the vaccine. The fusion gene gapdh gene and eta1 gene was amplified by PCR. The target gene was inserted into the expression vector p ET-30a (+) and transformed into E. coli BL21 (DE3) to express the fusion protein. SDS- -PAGE to detect the expression of the fusion protein. The fusion protein was purified by Ni-Sephadex G-100 and Sephadex G-100. Finally, the purified fusion protein was verified by Western-blot. The results showed that the fusion protein was mainly expressed in inclusion bodies. The content of the fusion protein was up to 73.6% after the cell pellet was broken, and the purity of the purified fusion protein was as high as 90%. The purified protein was verified by Western-blot. The above results show that the expression vector was successfully constructed and the protein of interest was abundantly expressed in E. coli.