| 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。 |
| 关键词: 米非司酮 诱导表达 调控 真核表达载体 |
| DOI：10.3724/SP.J.1008.2008.00371 |
| 投稿时间：2007-07-10 |
| 基金项目:国家自然科学基金（30571830）. |
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| Construction and identification of a mifepristone inducible eukaryotic expression vector |
| CHEN Jian1，2,XUE Xu-chao2*,FANG Guo-en2,SU Chang-qing3,QIAN Qi-jun3 |
| (1.Department of Oncosurgery,No.81 Hospital,PLA Nanjing Military Area Command,Nanjing 210002 ,China; 2.Department of General Surgery,Changhai Hospital,Second Military Medical University,Shanghai 200433;3.Laboratory of Viral and Gene Therapy, Eastern Hepatobiliary Hospital,Second Military Medical University,Shanghai 200438) |
| 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. |
| Key words: mifepristone inducible expression regulation eukaryotic expression vector |
