摘要
为了构建绿色荧光蛋白AcGFP1基因的原核表达载体pET32a-AcGFP1,并诱导使其在大肠杆菌中高效表达,本研究以pIRES-AcGFP1质粒为模板,采用PCR技术特异性扩增绿色荧光蛋白AcGFP1基因,通过酶切和连接使其与原核表达载体pET32a(+)构成重组子,经PCR、酶切和测序鉴定后,重组质粒转化大肠杆菌Rosetta(DE3),用不同浓度的异丙基硫代半乳糖苷(IPTG)诱导表达绿色荧光蛋白,经15%SDS-PAGE鉴定,结果显示:重组质粒pET32a-AcGFP1中的AcGFP1序列与Clontech公司的pIRES-AcGFP1质粒的AcGFP1序列完全一致,表明本实验已成功构建了含有绿色荧光蛋白基因AcGFP1的原核表达质粒。此外,pET32a-AcGFP1转化Rosetta(DE3),经不同浓度的IPTG诱导,SDS-PAGE检测均获得高效表达,为今后构建pET32a(+)-TAT-AcGFP1融合表达载体以及深入研究TAT的细胞膜穿透作用的机制奠定基础。
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-15-D-thiogalactopyranoside （IPTG） and de- tected 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 pE]32a-AcGFP1 plasmids transformed in E. coli Rosetta （DE3） were high efficient expression after inducing by different concentrations of IPTG. Re- suits above will provide a basis on constructing pE332a （ ＋ ） -TAT-AcGFP1 fusion expression vector and studying deeply mechanism of TAT cell membrane penetration.
出处
《云南农业大学学报（自然科学版）》
CAS
CSCD
北大核心
2012年第6期820-825,共6页
Journal of Yunnan Agricultural University:Natural Science
基金
云南农业职业技术学院科学研究与技术开发重点项目(YNAVC201116)
云南省教育厅科学研究基金项目(2011C191)
国家自然科学基金项目(31160439)
关键词
绿色荧光蛋白
基因克隆
报告基因
原核表达
green fluorescent protein
gene cloning
reporter gene
prokaryotic expression
