尼罗罗非鱼Galectin-3基因的原核表达与条件优化

Prokaryotic Expression and Condition Optimization of Galectin-3 Gene in Oreochromis

【目的】对Galectin-3蛋白进行纯化,优化诱导相关表达条件,为大量获取罗非鱼Galectin-3蛋白提供方案。【方法】根据NCBI上已公布的罗非鱼(Oreochromis mossambicus)Galectin-3基因序列,设计多对带EcoRI和Xhol酶切位点的引物,筛选出良性扩增引物,对罗非鱼cDNA经进行聚合酶链式反应(PCR)扩增、限制性快切酶双酶切、T4连接酶连接等步骤,构建罗非鱼Galectin-3基因的原核表达质粒pGEX-4T-Galectin-3,将重组质粒导入大肠杆菌BL21中,进行Galectin-3重组蛋白的表达。并比较不同的诱导温度、诱导时间、IPTG浓度条件下的表达效果,对Galectin-3蛋白表达最优条件进行筛选。【结果】构建了罗非鱼Galectin-3原核表达质粒,进行Galectin-3重组蛋白后发现37℃下,0.4mmol/L浓度的IPTG诱导5h即可诱导Galectin-3重组蛋白高效表达。免疫印迹结果显示,经蛋白纯化柱纯化后的pGEX-4T-Galectin-3重组蛋白可与GST-Tag单克隆抗体发生特异性反应,进而表明表达的重组蛋白是罗非鱼的Galectin-3蛋白。【结论】本研究成功构建了罗非鱼Galectin-3基因的原核表达载体,并确定了Galectin-3融合蛋白最适的表达条件:37℃下,0.4mmol/L浓度的IPTG诱导5h。

【Objective】The prokaryotic expression and expression conditions of galectin-3 gene were optimized to obtain a large amount of proteins, laying a foundation for further preparation of monoclonal antibodies.【Methods 】Based on the published sequence of Galicon (Oreochromis mossambicus) Galectin-3 gene on NCBI, several pairs of primers with EcoRI and Xhol restriction sites were designed to screen for benign amplification primers, and the tilapia cDNA was polymerase-chained. The prokaryotic expression plasmid pGEX-4T-Galectin-3 of tilapia Galectin-3 gene was constructed by reaction (PCR) amplification, restriction enzyme digestion and T4 ligase ligation, and the recombinant plasmid was introduced into E. coli BL21. In the expression of Galectin-3 recombinant protein, we compared the expression effects of different induction temperature, induction time and IPTG concentration, and screened the optimal conditions for Galectin-3 protein expression.【Results】pGEX-4T-Galectin-3 was successfully constructed and the Galectin-3 recombinant protein was expressed. It was found that the high expression of Galectin-3 recombinant protein was induced by IPTG at a concentration of 0.4 mmol/L for 5 h at 37 ℃. Western blot results showed that the recombinant protein pGEX-4T-Galectin-3 purified by protein purification column could specifically react with GST-Tag monoclonal antibody, indicating that the expressed recombinant protein is Galectin-3 protein of tilapia.【Conclusion】In this study, the prokaryotic expression vector of the tilapia Galectin-3 gene was successfully constructed, and the optimal expression conditions of the Galectin-3 fusion protein were determined. The highest concentration was obtained by inducing IPTG at a concentration of 0.4 mmol/L for 5 h at 37 ℃.