摘要
构建可经RU486诱导表达载体,并证实其对基因表达的调控作用。通过分子生物学技术,改造了含有GLP65反式作用调控因子和GAL4杂合启动子的PRS质粒。PCR扩增BGHpolyA片段,并引入需要的酶切位点。在GLP65调控区上游添加了hCMV启动子,在GAL4杂合启动子下游加入了荧光素酶报告基因。同时,为减少两个转录单元之间的潜在干扰,加入了1.2kb的小鸡β珠蛋白绝缘子。经PCR和限制性酶切及测序证实了载体的正确性。在体外转染HEK293细胞后,运用双荧光素酶报告基因技术鉴定了该系统的调控能力。加入诱导剂RU486后,可以诱导表达荧光素酶,并在一定范围内两者呈正比,最高可以实现荧光素酶的40余倍的表达,而没有RU486时,几乎没有报告基因的表达,表明RU486诱导调控载体构建成功,可实现对目的基因的表达时间和表达水平的精确调控,为进一步的基因调控研究和基因治疗提供了良好的工具。
To construct a expression vector induction by RU486 （mifepristone） and confirm the regulatable effect in v/tro. The PRS plasmid vector encoding a GLP5 chimeric regulator and GAL4 hybrid promoter was modified with molecular biological methods. The BGHPOlyA fragment was amplified by PCR technique and introduced several enzyme cutting sites. A hCMV promoter replaced the TI＇R promoter to control the GLP5 regulator, and luciferase reporter gene was inserted into downstream of the GAL4 promoter. To minimize any potential interference, the two transcriptional elements were spaced with a 1.2kb chicken beta-globin insulator. The recombination vector was identified by different restriction endonuclease reactions, sequencing analysis and PCR assay. Dual-Luciferase Reporter Assay was used to demonstrate the activation of this regulatable vector after cotransfection with pRL-TK in HEK293 cells. Forty-eight hours after transfection, cells were harvested to determine luciferase activity by illuminometer, In the presence of RU486 a 40-fold maximum activation of the luciferase reporter gene was observed in cultured cells, whereas in the absence of RU486, no significant activation was observed. There was a positive correlation between the luciferase activation and RU486 concentration in a definite range. The results showed that the RU486-inducible regulatory vector was successfully constructed, which can be used to regulate the expression level of genes of interest in appropriate time by the inducer RU486. This inducible expression vector provides a platform for the research of gene regulation and gene therapy.
出处
《中国生物工程杂志》
CAS
CSCD
北大核心
2007年第6期1-5,共5页
China Biotechnology
基金
国家自然科学基金资助项目(30571830)
