摘要
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.
出处
《天津医科大学学报》
2008年第1期1-4,21,共5页
Journal of Tianjin Medical University
基金
"863"国家高技术研究发展计划(2007AA02Z115)
国家教育部新世纪人才支持计划(NCET-04-0245)
国家自然科学基金(30670441
30300070)
天津市科委应用基础研究重点项目(07JCZD-JC07300)
天津市科委国际合作项目(05YFGHHZ01300)