基于Tet-on系统的RFP基因表达载体构建及功能验证

发布时间：2018-04-30 19:17

本文选题：红色荧光蛋白基因 + Tet-on系统　；参考：《西北农林科技大学学报(自然科学版)》2017年08期

【摘要】：【目的】构建四环素诱导表达调控系统的表达载体,并对其功能进行验证。【方法】利用pTRE-tight和pdsred-Express2-N1构建带有红色荧光蛋白(red fluorescence protein,RFP)基因的载体pTRE-DSred,用其与另一载体pTet-on共转染293T细胞,并加入不同质量浓度(0,0.1,1,10μg/mL)的强力霉素(doxycycline,DOX)诱导,以未经质粒转染的293T细胞为对照组,在荧光显微镜下观察RFP基因的表达情况。【结果】成功构建了pTRE-DSred重组质粒,将其与pTet-on质粒共转染293T细胞,在细胞培养液中分别添加0.1,1,10μg/mL DOX,48h后均有红色荧光蛋白的表达,但10μg/mL DOX组红色荧光蛋白的表达水平明显低于前2个组;0μg/mL DOX诱导组中也有红色荧光蛋白表达,但水平极低;在未转染质粒对照组中未观察到红色荧光蛋白的表达。RT-PCR结果显示,pTREDsred和pTet-on共转染组均检测到RFP基因的转录。【结论】成功构建了Tet-on系统RFP基因表达载体,适宜质量浓度的DOX诱导有助于目的基因的高水平表达,但在不添加DOX诱导剂时,该Tet-on系统存在目的基因低渗漏表达现象。
[Abstract]:[objective] to construct the expression vector of tetracycline inducible expression regulatory system and verify its function. [methods] using pTRE-tight and pdsred-Express2-N1 to construct the vector pTRE-DSredwith red fluorescent protein fluorescence pdsred-Express2-N1 gene and co-transfect 293T cells with another vector pTet-on. Doxycycline dox (doxycycline dox) was induced by 10 渭 g / mL doxycycline (doxycycline dox). The expression of RFP gene in 293T cells was observed under fluorescence microscope. [results] Recombinant pTRE-DSred plasmid was constructed successfully. It was co-transfected with pTet-on plasmid into 293T cells, and the red fluorescent protein was expressed in the cell culture medium after 48 hours after the addition of 0.1 ~ 10 渭 g/mL dox to the cell culture medium. However, the expression level of red fluorescent protein in 10 渭 g/mL DOX group was significantly lower than that in 0 渭 g/mL DOX induced group, but the level was very low. The expression of red fluorescent protein was not observed in the untransfected plasmid control group. RT-PCR results showed that both pTREDsred and pTet-on cotransfected group detected the transcription of RFP gene. [conclusion] the expression vector of RFP gene of Tet-on system was successfully constructed. The optimal concentration of DOX induces high level expression of the target gene, but the expression of the target gene is low in the Tet-on system without adding DOX inducer.
【作者单位】：华南农业大学动物科学学院广东省农业动物基因组学与分子育种重点实验室;河南牧业经济学院动物科技学院;
【基金】：国家“973”计划专项(2011CB944202) 河南牧业经济学院博士科研启动基金项目(51000788)
【分类号】：Q78

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