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目的 构建新型AFP顺式作用元件调控的基因表达载体 ,检测该调控元件的特异性和可调控性。方法 PCR扩增截短的AFP基因启动子、增强子 ,将上述片段与含有报告基因强化绿色荧光蛋白基因的载体pEGFP 1的多克隆位点连接 ,构建成为AFP基因顺式作用元件调控的肝癌特异性EGFP表达载体 (pEGFP 1 EP)。用脂质体法将表达载体转染表达或不表达AFP的肿瘤细胞系 ,荧光显微镜观测重组AFP顺式作用元件的启动活性 ,并用流式细胞术检测全反式视黄酸对它的抑制作用。结果 成功地将AFP基因启动子、增强子克隆到报告基因载体pEGFP 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 ,1× 10 -7M全反式视黄酸对其活性有明显的抑制。结论 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 new gene expression vector regulated by cis-acting element of AFP to test its specificity and regulatability. Methods The promoter and enhancer of truncated AFP gene were amplified by PCR. The fragment was ligated with the multiple cloning site of vector pEGFP 1 containing EGFP gene and constructed into a hepatocarcinoma regulated by the cis-acting element of AFP gene Sex EGFP expression vector (pEGFP 1 EP). The expression vector was transfected into tumor cell lines expressing or not expressing AFP by liposome method. The activation activity of cis-acting element of recombinant AFP was observed under a fluorescence microscope and the inhibitory effect of all-trans retinoic acid on it was detected by flow cytometry . Results AFP gene promoter and enhancer were successfully cloned into the multi-cloning site of reporter gene vector pEGFP 1. The results of restriction enzyme digestion and DNA sequence analysis showed that EGFP was specifically expressed in AFP positive hepatocarcinoma cells by 1 × 10 -7M all-trans retinoic acid significantly inhibited its activity. Conclusions AFP cis-acting gene modified gene therapy vector specifically regulates the expression of the target gene at the level of gene transcription, which lays the foundation for further treatment of hepatocellular carcinoma gene therapy vector.