猪表皮生长因子的原核表达及纯化

Expression and purification of recombinant porcine epidermal growth factor

[目的]:提高重组猪表皮生长因子(pEGF)的表达量和纯化量,为后续研究pEGF对仔猪生长发育的影响奠定基础。[方法]:将人工合成的pEGF基因克隆至pEGF4T-1载体,转化至大肠杆菌Rossetta(DE3)中,得到重组菌株Rossetta(DE3)-pGEX-pEGF,IPTG诱导表达,进行表达条件的优化,用GST-tag亲和层析纯化方法进行纯化。[结果]:pEGF基因大小为159 bp,融合表达蛋白分子量为32k Da,最佳诱导表达条件为IPTG浓度0.01mmol/L、诱导温度15℃和诱导时间24h。流穿液和洗涤液中目的蛋白含量极少,洗脱液得到了高纯度的GST-pEGF融合蛋白,纯化较为成功。[结论]:成功构建了pEGF表达菌株,得到了最优的诱导表达条件和最优的纯化工艺,为pEGF相关产品的开发以及应用奠定了基础。

[Objective]:To improve the expression and purification amount of recombinant porcine epidermal growth factor(pEGF),and lay a foundation for the follow-up study on the effects of pEGF on the growth and development of piglets.[Methods]:The recombinant strain Rosseta(DE3)-p GEX-pEGF was cloned into pGEX4T-1 vector and transformed into Escherichia coli Rossetta(DE3).The recombinant strain Rosseta(DE3)-p GEX-p EGF was induced to express by IPTG.The expression conditions were optimized and purified by GST-Tag affinity chromatography.[Results]:pEGF gene size was 159 bp,the molecular weight of the fusion expression protein was 32k Da,and the optimal induction conditions were IPTG concentration of 0.01mmol/L,induction temperature of 15℃and induction time of 24h.GST-pEGFF fusion protein was obtained in the eluent with high purity,and the purification was successful.[Conclusion]:The p EGF expressing strain was successfully constructed,and the optimal expression conditions and purification process were obtained,which laid a foundation for the development and application of pEGF related products.