可经米非司酮诱导的真核表达载体的构建与鉴定  

Construction and identification of a mifepristone inducible eukaryotic expression vector

作　　者：陈坚[1] 薛绪潮 方国恩[2] 苏长青[3] 钱其军[3] 

机构地区：[1]解放军第81医院肿瘤外科,南京210002 [2]第二军医大学长海医院普通外科,上海200433 [3]第二军医大学东方肝胆外科医院病毒基因治疗实验室,上海200438

出　　处：《第二军医大学学报》2008年第4期371-375,共5页Academic Journal of Second Military Medical University

基　　金：国家自然科学基金(30571830)~~

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Lipo- fectamine2000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^(-10)mol/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。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^-6mol/ 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.

关 键 词：米非司酮 诱导表达 调控 真核表达载体 

分 类 号：Q78[生物学—分子生物学]