鲑鱼甲病毒E1基因高保守区融合表达及免疫特性的分析

Prokaryotic expression and analysis of a recombinant fusion protein of highly conserved region of salmonid alphavirus E1 gene

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。

Highly conservative sequence of salmonid alphavirus E1 gene（ 702 bp） was synthesized according to sequences of E1 gene of SAV1,SAV2 and SAV3 published in Gen Bank. It was cloned into prokaryotic expression vector p Cold TF to construct recombinant plasmid. The recombinant plasmid was transformed into E. coli BL21 cells. The expression of recombinant plasmid p Cold TF in E. coli BL21 was induced using IPTG at 1. 0 mmol / L. The target protein of 95 k D was obtained and confirmed by SDS-PAGE and Western blot. The recombinant protein was purified by nickelionaffinity chromatography,and antiserum was produced. Indirect El ISA showed the antiserum against E1 protein had OD values at least twice that obtained for the negative control serum at a dilution of 1∶ 25 600. IFA showed the antiserum against E1 protein reacted specifically with SAV. The results indicated that the recombinant E1 protein was immunogenical and antigenical,which established a foundation for further study on detection method of SAV.