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目的:构建缺氧诱导的AFP基因启动子(HRE-AFPp)调控的基因表达载体,并检测该调控元件的特异性和对缺氧诱导的反应性。方法:用PCR方法从人类基因组中扩增VEGF基因缺氧反应元件(HRE)和AFP基因启动子(AFPp),将上述片段与含有报告基因(强化绿色荧光蛋白基因)载体pEGFP-1的多克隆位点连接,构建成为缺氧诱导的AFP基因启动子调控的基因表达载体(pEGFP-[HRE]AFPp)。用脂质体法将表达载体转染表达或不表达AFP的细胞系,G418筛选阳性克隆,荧光显微镜观测重组AFPp的活性,并用流式细胞术检测缺氧诱导是否对其有调控作用。结果:成功地将HRE、AFPp克隆到报告基因载体pWGFP-1的多克隆位点,酶切鉴定和DNA序列分析无误,荧光显微镜观测证实EGFP能在AFP阳性肝癌细胞特异性表达,流式细胞术检测证实,16 h缺氧诱导能增强EGFP的表达(P<0．01)。结论:缺氧诱导的AFP顺式作用元件修饰的基因治疗载体,在基因转录水平特异性调控目的基因的表达,为下一步将其作为肝癌基因治疗载体奠定了基础。
Objective: To construct a hypoxia-inducible AFP gene promoter (HRE-AFPp) gene expression vector and to detect the specificity of the regulatory elements and hypoxia-induced reactivity. Methods: The VEGF gene hypoxia response element (HRE) and AFP gene promoter (AFPp) were amplified from the human genome by PCR. The above fragment was cloned into a polyclonal vector containing the reporter gene (enhanced green fluorescent protein) pEGFP-1 Site to construct a gene expression vector (pEGFP- [HRE] AFPp) which is hypoxia-inducible AFP gene promoter. The expression vector was transfected into the cell lines expressing or not expressing AFP by lipofectamine. The positive clones were screened by G418 and the activity of recombinant AFPp was observed by fluorescence microscopy. Flow cytometry was used to detect whether hypoxia induced its regulation. Results: HRE and AFPp were successfully cloned into the multi-cloning site of reporter gene vector pWGFP-1. The restriction enzyme analysis and DNA sequence analysis were correct. Fluorescence microscopy confirmed that EGFP was specifically expressed in AFP positive hepatoma cells. Flow cytometry Test confirmed that, 16 h hypoxia can enhance the expression of EGFP (P <0.01). CONCLUSION: Hypoxia-induced AFP cis-acting gene modified gene therapy vector specifically regulates the expression of the target gene at the level of gene transcription, which will lay the foundation for further treatment of hepatocellular carcinoma gene therapy.