| 叶玲玲李世崇刘红刘兴茂何文俊陈昭烈.一种基于三顺反子表达载体的生物素诱导表达系统的建立[J].,2011,11(8):1404-1408 |
| 一种基于三顺反子表达载体的生物素诱导表达系统的建立 |
| Establishment of a Biotin-inducible Expression System Basedon Tricistronic Expression Vector |
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| DOI： |
| 中文关键词: 生物素 三顺反子 哺乳动物细胞 诱导表达系统 EGFP |
| 英文关键词: Biotin Tricistronic Mammalian cells Inducibleexpression system EGFP |
| 基金项目:国家“重大新药创制”科技重大专项（No. 2009ZX09503- 011） |
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| 中文摘要: |
| 目的：建立一种以无毒的生物素为诱导剂的哺乳动物细胞诱导表达系统。方法: 通过基因组PCR 或重叠延伸PCR 获得该系
统的三个基本构件：大肠杆菌生物素连接酶（BirA）、链亲和素- 四环素依赖的抑制因子融合蛋白（SA-TetR） 和生物素化信号
-VP16 转录激活结构域融合蛋白（Avitag-VP16）。将上述基因连入三顺反子表达载体，与响应载体一起共转染293f 细胞，以EGFP
为报告基因，检测EGFP 荧光强度随培养体系中生物素浓度变化的情况。结果: 随着生物素浓度的增加，目的基因表达出现
OFF-ON-OFF 的变化，诱导状态下的EGFP 荧光强度约为抑制状态下的3 倍。结论: 可通过调节生物素浓度对目的基因的表达进
行可逆的调节，该系统是一种有应用前景的诱导表达系统。 |
| 英文摘要: |
| Objective: To establish a mammalian inducible expression system with the non-toxic biotin as an inducer. Methods:
This system comprised three key components: E. coli biotin ligase (BirA), streptavidin-tetracyclin repressor (SA-TetR) and biotinylation
signal-VP16 transactivation domain (AT-VP16). The three genes were introduced into a tricistronic expression vector, and co-transfected
293f cells together with the response vector. The EGFP taken for a reporter gene, EGFP fluorescent intensities were detected under
different biotin concentrations. Results: With the increasing of biotin concentrations, EGFP displayed sequential OFF-ON-OFF
expression profiles, the fluorescence intensity of EGFP in the induced state was about 3 times that in the inhibited state. Conclusion: The
expression of the target gene could be reversibly regulated by adjusting the concentration of biotin . This is a promising inducible
expression system. |
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