高表达miR-122腺病毒载体构建与功能检测

Construction and identification of miR-122 expressing recombinant adenovirus

利用改造后的腺病毒表达载体pDC312-cmv,构建含有8个miR-122表达框的重组腺病毒载体p-8miR-122,通过RT-PCR、ELISA、Sourthern blot验证重组载体高表达miR-122水平及抗乙肝病毒活性的能力.结果表明:重组腺病毒载体p-8miR-122构建成功且可高表达miR-122.并且,为鉴定重组腺病毒高表达miR-122水平及抗乙肝病毒活性的能力,本文还分别对空载体pDC312-cmv和重组腺病毒载体p-8miR-122进行腺病毒包装、扩增、纯化和TCID50滴度检测,其结果表明:纯化后的对照腺病毒和重组腺病毒滴度分别高达3×1010IU/mL和1×1011IU/mL,重组腺病毒与对照病毒相比具有miR-122高表达和抗乙肝病毒活性的能力.

To construct miR-122 overexpression adenovirus vector,gene including eight miR-122 precursor fragments was inserted into recombinant construct pDC312-cmv.Abilities of miR-122 overexpression and anti-HBV activities by the recombinant vector p-8miR-122 were analyzed by RT-PCR、ELISA and Sourthern blot.Through adenovirus packaging、amplification、purification and TCID50 titer detection,the titers of pDC312-cmv and p-8miR-122 adenovirus were 3×10^10IU/mL and 1×10^11IU/mL.Adevirus p-8miR-122 had the abilities of miR-122 overepression and anti-HBV activities.