携带米非司酮可调控β内啡肽基因腺病毒的构建及鉴定

Construction and identification of adenovirus carrying human β-endorphin genes regulable by mifepristoneinducible system

目的构建携带米非司酮（RU486）可调控β内啡肽（β—EP）基因的腺病毒（Ad—RUNEP）。方法构建携带RU486可调控β-EP基因的腺病毒穿梭质粒pDC312-RUNEP，重组得到Ad—RUNEP后进行扩增、纯化和滴度测定，并分别在载体水平和病毒水平进行验证。Ad—RUNEP感染人皮肤癌上皮A431细胞株24h后，将A431细胞株随机分为6个不同浓度RU486组（R0-5组），每组5孔，均加入RU486诱导β—EP表达，终浓度分别为0、10^-10、10^-9、10^-8、10^-7和10^-6mol／L，应用RU486孵育48h后更换为不含RU486的培养液，于开始RU486孵育的第1天、第2天和第4天取培养液，离心后收集上清液，采用ELISA法测定上清液β—EP浓度。结果经酶切鉴定表明RU486调控系统与β—EP均正确插入pDC312-RUNEP；Ad—RUNEP病毒滴度为2．25×10^10pfu／ml。与R0组比较，R1-5组β—EP浓度升高（P〈0．05）；与凡组比较，R1-5组β—EP浓度降低（P〈0．05）；与孵育第1天比较，第2天时p—EP浓度升高（P〈0．05）；与孵育第2天比较，第4天时β—EP浓度降低（P〈0．05）。结论成功构建了Ad—RUNEP，RU486对Ad—RUNEP合成β-EP具有良好的调控作用。

Objective To construct and identify adenovirus （Ad-RUNEP） carrying human β-endorphin （β-EP） genes which can be regulated by mifepristone （RU486）-inducible system and to evaluate the effects of different concentrations of RU486 on the transgene expression in adenovirus in vitro. Methods The shuttle plasmid pDC312-RUNEP carrying β-EP genes which can be regulated by RU486-inducible system was constructed and was combined with adenovirus to form a recombinant Ad-RUNEP using AdMAXTM system. The recombinant Ad- RUNEP was then amplified and purified. The titers of the adenovirus were determined and the adenovirus vector was checked. After being infected by Ad-RUNEP for24 h, A431 cell line was incubated in liquid culture media containing RU486 0, 10^-10, 10^-9 , 10^-8 , 10^-7 , and 10^-6 mol/L respectively （group R0-5 ） for 48h, and was then transferred to liquid culture media containing no RU486. The liquid culture media were obtained on 1st, 2nd and 4th day of incubation and centrifuged. The supernatant was collected for determination of β-EP concentration by ELISA. Results The analysis of enzyme-incision demonstrated that RU486 regulating system and β-EP were cloned directly into pDC312-RUNEP. The titer of Ad-RUNEP was 2.25 × 10^10 pfu/ml. The expression of β-EP was significantly higher in group 1-5 than in group R0 and was significantly lower in group R1-3 than in group R4 （ P 〈 0.05）. The expression of β-EP was significantly higher on 2nd than on 1st and 4th day. Conclusion The adenovirus carrying β-EP genes which can be regulated by RU486 （Ad-RUNEP） was successfully constructed.