可经米非司酮诱导的真核表达载体的构建与鉴定
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国家自然科学基金(30571830).


Construction and identification of a mifepristone inducible eukaryotic expression vector
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Supported by National Natural Science Foundation of China(30571830).

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    摘要:

    目的:构建一种可经米非司酮诱导的真核表达载体,并用荧光素酶报告基因鉴定其调控作用。方法:利用分子生物学技术,将萤火虫荧光素酶LUC基因和启动子,以及米非司酮调控系统构建成单一的质粒载体pDC-RULUC,为减少两个转录单元之间的潜在干扰,加入了1.2 kb的绝缘子。通过PCR扩增和限制性酶切及测序分析鉴定载体的正确性。利用Lipofectamine2000试剂盒转染载体pDC-RULUC至体外培养的SW620细胞,将载体pGL3-Control和pGL3-Basic的转染分别设为阳性和阴性对照组,各组均同时转染pRL-TK载体作为内参照。实验组细胞在不同浓度(0、1×10-5、1×10-6、1×10-7、1×10-8、1×10-9、1×10-10mol/L)的米非司酮培养液中孵育48 h后,采用双荧光素酶报告基因测试系统检测荧光素酶相对活性。复孔组则予去除培养液中的米非司酮并继续孵育48 h后行荧光素酶相对活性检测。结果:PCR和限制性酶切及测序均证实了载体的正确性。加入诱导剂米非司酮后,荧光素酶的相对活性随着培养液中米非司酮的浓度增高而增加,当米非司酮浓度达到1×10-6 mol/L时,最高可以实现荧光素酶的50余倍的表达,而去除诱导剂米非司酮后,几乎检测不到报告基因的表达。结论:成功构建了新型的米非司酮诱导调控系统载体,可以在体外实现对目的基因表达的有效调控,为进一步的基因调控研究和基因治疗奠定了基础。

    Abstract:

    Objective:To construct a mifepristone(an oral nontoxic chemical)-inducible eukaryotic expression vector and to evaluate its regulatory effect in vitro using luciferase reporter gene.Methods: Vector pDC-RULUC,which contains firefly luciferase reporter gene,promoter and mifepristone-inducible system,was constructed by molecular biological methods.A 1.2 kb insulator was inserted to reduce the interference between two transcription units.The vector was verified by PCR,restriction enzyme digestion,and sequencing.pDC-RULUC was used to transfect SW620 cells using Lipofectamine2000.Cells transfected with pGL3-Control and pGL3-Basic were used as positive and negative controls,respectively.Cotransfectant with pRL-TK renilla luciferase reporter vector was used as internal control.Cells of experimental group were incubated for 48 h in presence of different concentrations of mifepristone after transfection and were harvested for luciferase assay by using the Dual-Luciferase Reporter Assay System.Half of the wells were replaced with fresh medium and were measured after another 48 h.Results: The recombined plasmid vector was identified by digestion with different enzyme restrictions,PCR and sequencing analysis.The relative activity increased with the increase of mifepristone concentration.When the concentration of mifepristone reached 1×10-6 mol/L,the relative activity increased to approximately 50 folds of the original.No significant luciferase activity was detected when the mifepristone was removed.Conclusion:We have successfully established mifepristone-regulated eukaryotic expression vector, which can be used for controllable gene expression in vitro,providing a way for gene regulation and gene therapy.

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  • 收稿日期:2007-07-10
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  • 在线发布日期: 2008-04-16
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