高效缺氧诱导表达载体的构建与鉴定

Construction and Identification of an Effective Hypoxia-Inducible Expression Vector

目的:构建缺氧诱导表达载体,以介导报告基因在缺氧环境下的特异、高效表达。方法:通过分子生物学方法,将鼠磷酸甘油酸激酶基因的缺氧应答元件(HRE)和最小CMV(mCMV)启动子重组,构建增强型绿色荧光蛋白(EGFP)或萤光素酶报告基因的可诱导载体;通过酶切鉴定和测序分析,证实载体获得正确的构建;将重组载体转染HeLa细胞,观察EGFP荧光强度并检测萤光素酶活性。结果:HRE/mCMV启动子调控的报告基因载体具有特异和高效的诱导活性。结论:构建了可以进行特异和高效缺氧诱导的报告基因载体,为其进一步的开发和应用奠定了实验基础。

Objective： To construct hypoxia-inducible expression vector, which mediated reporter gene to express specially and efficiently in hypoxia environment. Methods： The molecular biology methods were used to construct EGFP or luciferase reporter gene vectors driven under HRE/mCMV reconstituting promoter, containing the murine phosphoglycerate kinase hypoxia response elements（HRE） and a minimal cytomegalovirus（mCMV） immediate-early promoter, which was verified by restriction enzyme digestion and sequence analysis. To determine the inducibility, HeLa cells transfected with the reporter gene vectors were visualized for EGFP expression or harvested for luciferase assay. Results： The constructed vector under HRE/mCMV promoter presented high hypoxia induction specially and efficiently. Conclusion： The hypoxia-inducible vector for special and efficient expression under HRE/mCMV promoter was successfully constructed, which could be exploited further.