新加坡石斑鱼虹彩病毒ORF086蛋白的原核表达、纯化及抗体制备

Prokaryotic expression,purification and antibody preparation of SGIV ORF086 protein

新加坡石斑鱼虹彩病毒(Singapore grouper iridovirus,SGIV)是导致石斑鱼养殖严重经济损失的主要病原体。本研究构建了含有新加坡石斑鱼虹彩病毒(Singapore grouper iridovirus,SGIV)ORF086基因的重组表达质粒载体pET32a-ORF086,将其转化到大肠杆菌中进行融合表达,经SDS-PAGE分析和Western blot鉴定,证实重组大肠杆菌融合表达了SGIV ORF086蛋白。经异丙基-β-D-硫代半乳糖苷(isoprophl-thio-β-D-galactopyranoside,IPTG)浓度、诱导温度、诱导时间等诱导表达条件的优化,确定在0.7 mmol/L IPTG、16℃诱导14 h时可溶性SGIV ORF086重组蛋白占重组蛋白的60%。经镍琼脂糖凝胶柱纯化重组蛋白,纯化度达95%以上。用纯化的SGIV ORF086蛋白免疫小鼠,获得了高效特异的SGIV ORF086抗血清。

Singapore grouper iridovirus （SGIV） is a major pathogen resulting in heavy economic losses to grouper aquaculture. In this study, recombinant expression vector pET32a-ORF086 inserted with SGIV ORF086 gene is transformed into E. coli BL21 （DE3）. SDS-PAGE and Western blot analysis confirm that the transformed E. coli BI21 can express SGIV ORF086 recombinant protein. The soluble recombinant protein is highly expressed under induction conditions of exposure to IPTG（0.7 mmol/L） at 16 ℃ for 14 h and successfully purified by Ni-IDA His-Bind affinity column with above 95% purity. The polyclonal antibody is prepared by immunizing mice with purified SGIV ORF086 protein and proved to be specific against SGIV ORF086 protein.