四环素诱导型单载体的构建、体外表达与鉴定

摘要

Currently used pTet-on-Advanced regulatory system contains two plasmids, and when it is applied to transgenic animals, two types of genetically modified animals are required to be established and hybridized. This results are in high cost and low efficiency. To address this question, this study aims to construct and functionally characterize tetracycline-inducible single plasmid system. The rtTA and EGFP genes were amplified by PCR from plasmid pTet-on-Advanced and pEGFP-N1, and then sub-cloned into the pTRE-Tight plasmid. The pTRE-Tight-rtTA -EGFP was transfected to fetal porcine fibroblast using liposome transfection. The expression of EGFP under different concentration of doxycyline (Dox)(0.0001,0.001, 0.01, 0.1 and 1 g/L) were assayed by fluorescence inverted microscope, as the results displayed at 48 h after transfection, there were no expressed green fluorescent in the cells before Dox added, and there were still no expressed green fluorescent in the cells under the concentration of Dox were 0.0001, 0.001, 0.01 and 0.1 g/L until the concentration reached 1 g/L. This phenomenon revealed that the regulation effect of Dox was stimulated with the concentration of 1 g/L. The results showed that the recombinant vector pTRE-Tight-rtTA-EGFP which induced with Tet-on was successfully constructed, and its expression could be induced in fetal porcine fibroblast by Dox. This study provides a novel tool for quantitative transgene expression, and also produces important experimental data to prepare transgenic animals under controlled expression.%pTet-on-Advanced双载体表达系统应用于转基因动物研制,需要建立两种动物品系,经过杂交和筛选,才有可能得到转基因个体,操作繁琐,转化效率低.本研究在改良的四环素(Tet-on)基因可调控系统基础上,通过PCR的方法,分别从pTet-on-Advanced质粒和pEGFP-N1质粒中扩增rtTA和EGFP基因,测序正确后,将rtTA和EGFP基因亚克隆入pTRE-Tight载体中,构建四环素pTRE-Tight-rtTA -EGFP单载体表达系统,然后采用脂质体法,将新构建的单载体表达体系pTRE-Tight-rtTA -EGFP转染猪胎儿成纤维细胞,以不同浓度(0.0001、0.001、0.01、0.1和1g/L)强力霉素(Dox)诱导,通过荧光显微镜检测EGFP 的表达量.结果显示,转染48 h后,未加Dox无绿色荧光表达；1g/L Dos诱导可以观察到绿色荧光；Dox浓度分别为0.0001、0.001、0.01和0.1 g/L未观察到绿色荧光.表明l g/L Dox才能激发调控作用.本研究成功构建pTRE-Tight-rt TA-EGFP新型可控性真核表达载体,该载体在细胞中的表达受Dox的调控,能正常发挥作用.研究结果为转基因的定量表达提供了新的技术平台,也为将构建的单载体用于表达可控的转基因动物研究提供了重要科学依据.