低氧启动重组腺相关病毒的构建

目的构建低氧启动rAAV,介导目的基因仅在低氧时特异性高效表达.方法通过分子生物学的方法,分别构建pGL3-6 HRE-CMV-mp和pGL3-6 HRE-CMV-mp-WPRE载体,比较2种低氧启动子的特异性和在低氧时的启动能力.选择最佳的低氧启动子构建低氧启动rAAV载体.采用磷酸钙和氯仿-PEG8000/NaCl-氯仿法构建低氧启动rAAV病毒.rAAV病毒蛋白电泳和EGFP报告基因验证低氧启动rAAV病毒的构建和介导目的基因在低氧时的表达. 结果HRE-CMV-mp-WPRE是最佳的低氧启动子,在低氧时高效、特异的表达目的基因.低氧启动rAAV仅在低氧时介导报告基因EGFP的表达.采用6 HRE-CMV-mp-WPRE启动子的rAAV具有良好的低氧特异性和高效启动能力.结论采用最佳的低氧启动子,本研究实现了低氧启动rAAV的构建.

Construction of hypoxia-specific recombinant adeno-associated virus

Objective To construct hypoxia-inducible recombinant adeno-associated virus (rAAV), which mediates target gene to express specially and efficiently in hypoxia environment. Methods Molecular biochemistry methods were used to construct pGL3-6 HRE-CMV-mp and pGL3-6 HRE-CMV-mp- WPRE vector, which were compared with each other in specificity and efficacy of hypoxia promoter. The better one was used to construct hypoxia-induced rAAV. Hypoxia specific rAAV was produced through calcium phosphate and chloroform-PEG8000/NaCl-chloroform method. rAAV protein was defined through SDS-PAGE protein electrophoresis. The ability of hypoxia specific rAAV to mediate target gene's expression in hypoxia environment was tested green fluorescent protein. Results 6 HRE-CMV-mp-WPRE was the better hypoxia promoter, which mediated target gene's expressing in hypoxia environment specially and efficiently. Hypoxia rAAV mediated EGFP expressing only in hypoxia environment. With 6 HRE-CMV-mp-WPRE promoter, rAAV presented high hypoxia specially and efficiently. Conclusion With the best hypoxia promoter, hypoxia specific rAAV was constructed in this experiment.