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根据Gen Bank中公布的鲑鱼甲病毒(salmonid alphavirus,SAV)SAV 1、SAV 2和SAV3三个基因型中E1基因,选择高保守序列702 bp(436-1137)合成基因,命名为SAV E1,将其克隆到原核表达载体p Cold TF中构建重组质粒。然后将重组质粒转化到大肠杆菌感受态细胞BL21中,经终浓度为1.0 mmol/L的IPTG诱导表达,SDSPAGE和Western blot鉴定,重组蛋白均获得了表达,表达E1重组蛋白约95 k D。用镍离子亲和层析柱纯化重组蛋白,制备抗血清。间接ELISA结果显示,鼠抗重组蛋白E1血清效价为1∶25 600;间接免疫荧光结果显示,鼠抗重组E1蛋白血清可与SAV发生特异反应,由此表明表达的E1重组蛋白具有良好的免疫原性和免疫反应性,为SAV检测方法的建立提供理论依据。
The 702 bp (436-1137) synthetic gene of high conserved sequence was selected based on the E1 gene of salmonid alphavirus (SAV) SAV 1, SAV 2 and SAV3 published in Gen Bank and named as SAV E1 It was cloned into prokaryotic expression vector p Cold TF to construct a recombinant plasmid. The recombinant plasmid was then transformed into E. coli BL21 and induced with IPTG at a final concentration of 1.0 mmol / L. The recombinant protein was expressed by SDSPAGE and Western blot, and the recombinant protein was expressed at about 95 kD. The recombinant protein was purified by nickel ion affinity chromatography to prepare antiserum. Indirect ELISA results showed that the serum titer of mouse anti-recombinant protein E1 was 1:25 600. Indirect immunofluorescence results showed that mouse anti-recombinant E1 protein serum reacted specifically with SAV, indicating that the expressed E1 recombinant protein has good immunity It provides a theoretical basis for the establishment of SAV detection methods.