云南农业大学学报2012，Vol.27Issue(6)：820-825,6.DOI:10.3969/j.issn.1004-390X(n).2012.06.009
绿色荧光蛋白基因原核表达质粒pET32a-AcGFP1的构建及其在大肠杆菌中的高效表达
Construction of Prokaryotic Expression Plasmid pET32a-AcGFP1 of Green Fluorescent Protein and Their High Efficient Expression in Escherichia Coli
摘要
Abstract
To construct prokaryotic expression vector pET32a-AcGFP1 and make it express efficiently in E. Coli cell, the AcGFP1 gene of green fluorescent protein was amplified from pIRES-AcGFP1 plas-mid with PCR method, and was inserted into prokaryotic expression vector pET32a ( + ) by restriction enzyme digestion. After identified by PCR, restriction enzyme digestion and sequencing, the AcGFP1 gene "was induced with different concentrations' isopropyl-β-D-thiogalaetopyranoside (IPTG) and detected on 15% SDS-PAGE. Results showed that the sequence of AcGFP1 gene in pET32a-AcGFP1 re-combinant plasmid is consistent with that AcGFP1 sequence in pIRES-AcGFP1 plasmid from Clontech Ltd., proving that the prokaryotic expression vector with AcGFP1 gene of green fluorescent protein is constructed successfully. Moreover, all of the pET32a-AcGFP1 plasmids transformed in E. Coli Rosetta (DE3 ) were high efficient expression after inducing by different concentrations of IPTG. Results above will provide a basis on constructing pET32a ( + ) -TAT-AcGFP1 fusion expression vector and studying deeply mechanism of TAT cell membrane penetration.关键词
绿色荧光蛋白/基因克隆/报告基因/原核表达Key words
green fluorescent protein/ gene cloning/ reporter gene/ prokaryotic expression分类
生物科学引用本文复制引用
霍海龙,赵跃,王锐,侯慧芳,李卫真,张永云,刘丽仙,王配,霍金龙..绿色荧光蛋白基因原核表达质粒pET32a-AcGFP1的构建及其在大肠杆菌中的高效表达[J].云南农业大学学报,2012,27(6):820-825,6.基金项目
云南农业职业技术学院科学研究与技术开发重点项目(YNAVC201116) （YNAVC201116）
云南省教育厅科学研究基金项目(2011C191) （2011C191）
国家自然科学基金项目(31160439). （31160439）
