Construction and identification of adenovirus carrying human β-endorphin genes regulable by mifepristone-inducible system / 中华麻醉学杂志

ABSTRACT

Objective To construct and identify adenovirag (Ad-RUNEP) carrying human B-endorphin(B-EP) genes which can be regulated by mifepfistone (RU486)-inducible system and to evaluate the effects of different concentrations of RU486 on the transgene expression in adenovirus in vitro.Methods The shuttle plagmid pDC312一RUNEP carrying B-EP genes which can be regulated by RU486-inducible system was constructed and was combined with adenovims 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 for 24 h,A431 cell line was incubated in liquid culture media containing RU4860,10-10,10-9,10-8,10-7,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 supematant Was collected for determination of B-EP concentration by ELISA.Results The analysis of enzyme-incision demonstrated that RU486 regulating system and B-EP were cloned directly into pDC312-RUNEP.The titer of Ad-RUNEP was 2.25×1010 pfuml.The expression of B-EP was significantly higher in group R1-s than in group R0 and was significantly lower in group R1-3 than in group R4 (P<O.05).The expression of B-EP wag significantly higher on 2nd than on 1st and 4th day.Conclusion The adenovims carrying B-EP genes which can be regulated by RU486 (Ad-RUNEP) Wag successfully constructed.