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基于RU486调控系统的重组腺病毒Ad-NRF2的构建及其体外分析

In vitro construction and confirmation of adenovirus containing a mifepristone (RU486)-inducible regulation system for NRF2 gene

摘要:

目的 构建RU486调控系统介导的重组腺病毒Ad-NRF2,并在H460细胞中进行表达,验证NRF2基因表达是否可调控及表达效率.方法 在腺病毒载体的基础上,引入携带RU486(米非司酮)诱导调控系统的Nrf2基因;用LUC基因及Dsred基因对RU486调控系统进行验证,并通过免疫印迹和RT-PCR技术检测Nrf2的表达情况.结果 LUC和Dsred的表达都随RU486呈剂量依赖性增加;Ad-RUNrf2腺病毒感染H460细胞后,通过RT-PCR及免疫印迹结果显示Nrf2的表达量随着RU486剂量的增加而增加,去除RU486后,Nrf2的表达也随之减弱.结论 携带RU486可调控式Nrf2基因腺病毒构建成功,并且在细胞内RU486能够调节Nrf2因子的表达.

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abstracts:

Objective To construct an adenovirus containing a mifepristone (RU486)-inducible regulation system for NRF2 gene, express the product in H460 cell and verify whether the mentioned system can control the gene expression and assess its efficiency. Methods A RU486-inducible regulation system for Nrf2 gene was introduced into an adenovirus. The confirmation was performed through the LUG and Dsred genes. And the expression pattern of Nrf2 at the viral level was examined by Western blot and RT-PCR (reverse transcription-polymerase chain reaction). Results The expressions of LUG and Dsred showed a rising trend with the incremental dose of RU486. After the transfection H460 cell with Ad-RUNrf2, the results of RT-PCR and Western blot demonstrated that the expression of Nrf2 was elevated with a rising dose of RU486. After the removal of RU486, the expression of Nrf2 was reduced. Conclusion The construction of an adenovirus carrying Nrf2 gene regulated by a RU486-inducible system is successful, and RU486 can adjust the cellular expression of Nrf2 factor. The LUG and the Dsred expression assumes the dosage dependence along with RU486 to increase; after the Ad-RUNrf2 infects the H460 cell, through RTPCR and Western the Blot result demonstrated that the expression of Nrf2 increases along with the RU486 dosage increases, after removing RU486, the Nrf2 expression is weaken. Showing the construction of the adenovirus carrying Nrf2 gene regulated by the mifepristone ( RU486 )-inducible system is successful, and RU486 can adjust the Nrf2 factor in the cell the expression.

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