尼罗罗非鱼Galectin-related protein B基因的原核表达与条件优化

Prokaryotic Expression and Condition Optimization of Galectin-related Protein B Gene in Nile Ti⁃lapia

Galectin-related protein B是一种缺乏半乳糖苷结合活性的半乳糖凝集素相关蛋白B。基于NCBI公布的尼罗罗非鱼(Oreochromis niloticus)Galectin-related protein B基因序列设计带酶切位点引物,构建原核表达载体pET-28a-GRPB,将重组质粒导入大肠杆菌,诱导重组质粒的蛋白表达,并对蛋白表达条件进行优化。结果表明:在28℃条件下,IPTG浓度为0.20mmol/L,诱导6h时,诱导重组蛋白的效果最佳;经Western-blot免疫印迹显示,纯化后的重组蛋白能与带HIS标签的单抗特异结合,条带单一,进一步证明该重组蛋白为Galec⁃tin-related protein B蛋白。

Galectin-related protein B is a galactose lectin-related protein B which lacks galactoside binding activity.In this study,primers with restriction site were designed based on the Galectin-related protein B gene sequence of Nile tilapia(Oreochromis niloticus)published by NCBI,and the prokaryotic expression vector pET-28a-GRPB,was constructed.The recombinant plasmid was introduced into E.coli to induce the expression of the recombinant plas⁃mid protein.The factors affecting the expression of the protein were optimized.The results showed that the concentra⁃tion of the recombinant protein reached a maximum under the induced time of 6h,the induced temperature of 28℃and concentration of IPTG of 0.20mmol/L.Western-blot blotting showed that the purified recombinant protein could specifically bind to the monoclonal antibody with HIS tag and had a single band,which further proved that the recom⁃binant protein was Galectin-related protein B protein.