含有RU486诱导系统的IL-2基因表达质粒的构建及功能鉴定

Construction and functional identification of interleukin-2 gene expression plasmid with RU486 inducible system

目的:构建含有RU486诱导系统的小鼠白细胞介素2(IL-2)基因表达质粒,研究RU486系统对IL-2基因表达的调控作用。方法:用限制性内切酶ClaⅠ处理含有RU486诱导系统的质粒pRS17和编码IL-2基因的质粒pUC57-IL-2,将IL-2基因插入到pRS17构建pRS-IL-2;通过PCR及限制性酶切鉴定重组质粒;将重组质粒体外转染SMMC-7721细胞,用不同浓度的RU486进行处理;将重组质粒通过水流动力学注射法注射到小鼠体内,腹腔注射RU486进行诱导;通过ELISA方法检测细胞上清及血清中IL-2基因的表达。结果:重组质粒pRS-IL-2经过限制性内切酶消化及PCR分析,显示了预期的片段。细胞在1×10-8mol·L-1的RU486诱导下,IL-2表达水平最高,是无RU486组的1.95倍(P<0.001)。注射质粒后,接受RU486诱导的小鼠血清中IL-2水平较诱导前升高320倍,而未接受RU486的小鼠血清未检测到IL-2的表达。结论:成功构建了含有RU486诱导系统的IL-2基因表达质粒,IL-2基因的表达依赖于诱导剂RU486的存在。

Objective To construct an interleukin-2 （IL-2） expression plasmid containing RU486 inducible system and study the regulatory effect of RU486 system on IL-2 gene expression.Methods The plasmid pRS17 containing an RU486 inducible system and plasmid pUC57-IL-2 coding mouse IL-2 gene were digested by restriction enzyme Cla Ⅰ,pRS-IL-2 was constructed by inserting IL-2 gene into pRS17.PCR and restriction enzymes were used to identify the recombinant plasmid.SMMC 7721 cells were treated with RU486 at different concentrations following transfection with recombinant plasmid.The recombinant plasmid was administered into the mice by hydrodynamic injection followed by intraperitoneal injection of RU486.The expressions of IL-2 gene in supernatant and serum were measured by using ELISA.Results By the restriction enzyme digestion and PCR analysis,the recombinant plasmid pRS-IL-2 showed the expected bands.The highest level of IL-2 was detected in the supernatant of cells treated with 10-8 mol·L-1 of RU486,which was 1.95-fold higher than that of the cells without RU486 （P0.001）.The serum IL-2 level in mice after stimulation of RU486 was 320-fold higher than before RU486 stimulation.IL-2 was undetectable in mice without RU486 injection.Conclusion The IL-2 gene expression plasmid containing RU486 inducible system is successfully constructed.The expression of IL-2 gene is dependent on RU486 presence.