EGF-SEA融合蛋白在大肠杆菌中的表达和纯化

Expression and purification of fusion protein EGF-SEA in E.coli

根据基因库中查到的金黄色葡萄球菌肠毒素A(SEA)基因序列和人体表皮生长因子(EGF)基因序列进行密码子优化,以适于大肠杆菌表达。人工合成SEA基因与EGF基因。将两目的基因克隆至原核表达载体pET22b中,经测序验证表明成功构建了重组表达质粒pET22b-EGF-SEA。将构建好的pET22b-EGF-SEA质粒转化大肠杆菌BL21(DE3),经IPTG诱导进行表达;SDS-PAGE分析表明融合基因EGF-SEA在大肠杆菌BL21(DE3)中以包涵体的形式得到了高效表达,产物相对分子质量约为44kDa,与理论值大小一致。包涵体经洗涤,变性、复性后用His.BindKit进行分离纯化,所得蛋白纯度≥95%。高纯度EGF-SEA融合蛋白的获得为进一步研究其生物学活性及肿瘤治疗奠定了基础。

The staphylococcal enterotoxin A （SEA） gene and epidermis growth factor（EGF） gene were obtained from GeneBank. Both of the gene codons were optimized and synthesized. After digested by appropriate restriction enzymes, SEA and EGF gene were successively cloned into expression vector pET22b. DNA sequencing verified that recombinant plasmid pET22b-EGF-SEA was constructed successfully, and then it was transformed into E. coli BL21 （DE3）. SDS- PAGE analysis revealed that after IPTG induction fusion protein EGF-SEA was over-expressed mainly in form of inclusion boty, and it had a molecular weight of about 44kDa in accordance with the theoretical value. After the inclusion boty was denatured with 8mol/L urea and then renatured by dialysis, His· Bind Buffer Kit was employed to purified the fusion protein. Finally, the protein purity could reach more than 95 %, witch laid a base for further study of the biological activities of EGF-SEA protein against tumors.