GST-G3BP Domain 1～5原核表达质粒的构建及其表达

The construction and expression of GST-G3BP Domain 1～5 fusion plasmid in procaryote

目的:构建GST-G3BP Domain1～5的融合表达质粒,并在大肠杆菌中稳定表达。方法:PCR扩增G3BP Domain1～5片段,利用限制性内切酶EcoRI和NotI将其连接至载体pGEX-4T-1,将连接产物转化大肠杆菌,挑取单菌落,提取质粒,经PCR和双酶切鉴定后测序。将转化正确的大肠杆菌过夜培养,IPTG诱导融合蛋白表达,immobilized Glutathione beads钓取目的蛋白。结果:G3BP Domain1～5片段均成功连接至载体pGEX-4T-1,在大肠杆菌BL21中可得到稳定表达的融合蛋白。结论:GST-G3BP Domain1～5的融合表达质粒构建成功。

Objective： To construct the fusion plasmid of GST-G3BP Domain lto 5 and let it express in E coli stably. Methods： G3BP Domain 1 to 5 fragments were obtained by PCR and were inserted into vector pGEX-4T-1 with EcoR I and Not I limited enzyme. The ligated vector was transformed into E coll. Chosen a single colony, the plasmid was. extracted and identified by double limited enzyme and PCR. The correctly ligated plasmid then was sent to sequence. The E coli with correct plasmid was cultured over night to induct the expression of the fusion protein by IPTG and pull down the fusion protein with immobilized Glutathione beads. Results： G3BP domain 1 to 5 fragments were correctly ligated with vector pGEX-4T-1, and the fusion protein could express in E coli stably. Conclusion： The fusion plasmid GST-G3BP Domain lto 5 are constructed successfully.