| 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(0，0.1，1，10 μg/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0.1，1，10 μg/mL DOX，48 h后均有红色荧光蛋白的表达，但10 μg/mL DOX组红色荧光蛋白的表达水平明显低于前2个组；0 μg/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，pTRE-Dsred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。 |
| 关键词: 红色荧光蛋白基因 Tet-on系统 强力霉素 载体构建 |
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| 基金项目: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） |
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| Construction and functional verification of Tet-on system of RFP gene expression vector |
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ZHANG Jingfeng,ZHU Kuanyou,XU Qiuliang,et al
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| Abstract: |
| 【Objective】This paper established the expression vector of tetracycline Tet-on system and verified its functions.【Method】A red fluorescent gene carrier pTRE DSred was constructed using the plasmids pTRE-tight and pdsred Express2-N1,and was transfected 293T cells together with plasmid pTet on.Then,doxycycline (DOX) with different concentrations (0,1,0.1,and 10 μg/mL) was added into the 293T culture medium to observe the expression of RFP using cultured 293T cells without plasmid as control.【Result】A carrier pTRE-DSred plasmid was constructed and it was used to transfect 293T cells with pTet-on plasmid.After adding 0.1,1,and 10 μg/mL DOX for 48 hours,expression of red fluorescent protein was obtained and the expression in 10 μg/mL DOX group was significantly lower than in the other two.The expression in 0 μg/mL DOX group was extremely low,and no expression was observed in the control group.【Conclusion】The Tet-on system RFP gene expression vector was constructed and adding proper DOX increased the expression. Low leakage phenomenon existed in the Tet-on system if no DOX was added. |
| Key words: RFP gene Tet-on system DOX the construction of the carrier |
