摘要
目的构建能在体外大量表达可溶性天然型增强绿色荧光蛋白(EGFP)的pET28a-EGFP原核表达载体。方法以质粒pEGFP-N1为模板扩增EGFP基因,EcoR I和Hind III双酶切EGFP基因序列和pET28a载体。T4 DNA连接酶连接,转化大肠埃希菌DH5α,提取载体,酶切和DNA测序鉴定。将pET28a-EGFP转化大肠埃希菌BL21(DE3),异丙基-β-D-硫代半乳糖苷(IPTG)诱导,观察荧光。体外翻译系统中加入重组pET28a-EGFP载体,孵育后观察荧光。免疫印迹法(Western blott)鉴定EGFP。结果测序证实pET28a-EGFP中的EGFP基因序列与GenBank中序列完全一致,荧光显微镜下观察到大肠埃希菌BL21(DE3)和体外翻译系统中强烈荧光,West-ern blot结果显示相对分子质量约为27 000的EGFP高效表达。结论成功构建了能在体内和体外稳定表达的可溶性天然型EGFP的pET28a-EGFP载体,为进一步体外筛选抑菌药物提供了可靠的报告载体。
Objective To construct the pET28a enhanced green fluorescent protein(EGFP)plasmid which can express the soluble native EGFP in cell-free translation system. Methods Using plasmid as template , full-length EGFP gene was amplified. The amplified EGFP gene and pET28a vector were cleaved by EcoR Ⅰ and Hind Ⅲ, then ligated after purifying. The ligated products were transformed into DH5α. The recombinant plasmid was extracted for further identification and sequencing. Escherichia Coli( E. coli) BL21 (DE3) was transformed with pET28α-EGFP, and induced by Isopropyl-beta-D-thiogalactoside(IPTG) for protein expression. The fluorescence was observed by fluorescence microscope. The recombinant pET28α-EGFP was added into the Cell-free translation system and incubated , the fluorescence was observed. EGFP was identified by Western blot. Results It was confirmed by DNA sequencing that the EGFP gene sequence in recombinant plasmid pET28α-EGFP was in accordance with the EGFP gene sequence in GenBank. The fluorescence was observed in E. coli BL21 (DE3)and cell-free translation system. Western bloting demonstrated that the high efficient expressed protein was native EGFP which molecular weight was about 27 000. Conclusions The recombinant plasmid pET28α-EGFP which can express the soluble native EGFP in E. coli BL21 (DE3) and Cell-free translation system is constructed successfully, it provides a reliable reporter vector to screening the bacteriostatic agents in vitro.
出处
《检验医学》
CAS
北大核心
2007年第2期146-149,共4页
Laboratory Medicine
关键词
增强绿色荧光蛋白
pET28a载体
重组载体
体外翻译系统
原核表达
Enhanced green fluorescent protein
pET28a vector
Recombinant plasmid
Cell-free translation system
Prokaryotic expression