摘要
目的:构建能在无细胞翻译系统中表达天然型增强绿色荧光蛋白(EGFP)的原核表达载体。方法:以质粒pEGFP-N1为模板扩增EGFP基因,NcoI和HindⅢ双酶切EGFP基因和pET28a载体。T4 DNA连接酶连接,转化E.coliDH5α,提取载体,酶切和DNA测序鉴定。正确重组的载体命名为pET28a-EGFP。在无细胞翻译系统中加入大抽后的pET28a-EGFP模板(0.6 mg/ml),孵育3 h。荧光显微镜观察荧光。Western blot鉴定EGFP。结果:测序证实重组EGFP基因序列与基因库中的序列完全一致,荧光显微镜下观察到无细胞翻译系统中强烈荧光,Western blot证实分子量约为27 kD的EGFP高效表达。结论:成功构建了能在无细胞翻译系统中高效表达可溶性天然型EGFP的pET28a-EGFP载体,为进一步建立荧光强度标准物奠定了基础。
Objective： To construct the prokaryotic expression plasmid that can express enhanced green fluorescent protein（EGFP） in a cell-free translation system. Methods： Utilizing plasmid as template, full-length EGFP gene was amplified. The pEGFP-N1 gene digested with NcoⅠ, Hind Ⅲ was ligated to pET28a vector and digested with the same restriction enzymes, then the resultant ligation product was transformed into E. coli DHSα. Positive clones were propagated and recombinant plasmids were extracted for further identification and sequencing. The sequence-confirmed recombinant plasmid was then given the name, pET28a-EGFP. The macroprepared pET28a-EGFP （0.6mg/ml） was added into the Cell-Free translation system,and incubated for 3 hours. The fluorescence was observed by the fluorescence microscopy. EGFP was identified by Western blotting. Results： DNA sequencing confirmed that the EGFP gene sequence in pET28a-EGFP was in accordance with that of GenBenk. The intensive fluorescence in cell-free translation system was seen under the fluorescence microscope. Western blotting demonstrated that expressed protein of about 27000 was native EGFP. Conclusion： The pET28a-EGFP which can efficiently express the soluble native EGFP in cell-free translation system was successfully constructed, which provided a basis to further establish a standard substance of fluorescence intensity.
出处
《江苏大学学报（医学版）》
CAS
2007年第3期225-227,231,共4页
Journal of Jiangsu University：Medicine Edition
关键词
增强绿色荧光蛋白(EGFP)
重组载体
无细胞翻译系统
原核表达
enhanced green fluorescent protein （EGFP）
recombinant plasmid
cell-free translation system
prokaryotic expression
