中文摘要：

目的 构建并选择肝癌特异性甲胎蛋白（AFP）基因启动元件。方法 采用PCR方法从小鼠肝脏克隆AFP基因增强子Ⅰ、抑制子和启动子，经过不同组合构建了两个真核表达载体：pEGFP-1-EP（含调控序列Ⅰ：增强子Ⅰ＋启动子）和pEGFP-1-ERP（含调控序列Ⅱ：增强子Ⅰ＋抑制子＋启动子）。通过瞬时转染，采用荧光显微镜观察和流式细胞仪检测所构建调控序列分别在小鼠肝癌细胞株Hepal-6、黑色素瘤细胞株B16和成纤维细胞株NIH3T3的活性。结果 所得AFP基因增强子Ⅰ、抑制子和启动子序列与Genebank中公布的相关序列-致；荧光显微镜下：pEGFP-1-EP和pEGFP-1-ERP转染Hepal-6细胞呈强阳性反应，pEGFP-1（空载体）转染Hepal-6和三种质粒转染的B16、NIH3T3细胞均呈阴性反应；流式细胞仪检测结果显示：pEGFP-1-EP转染Hepal-6阳性细胞率和平均荧光强度高于pEGFP-1-ERP转染Hepal-6细胞，两者阳性细胞率和平均荧光强度均高于pEGFP-1转染Hepal-6细胞；pEGFP-1转染Hepal-6和三种质粒转染B16、NIH3T3阳性细胞率和平均荧光强度均较低。结论 我们所构建小鼠AFP基因凋控序列Ⅰ和调控序列Ⅱ的启动活性均具有肝癌特异性，且前者高于后者，并选择调控序列Ⅰ在下一步肝癌特异性基因治疗中使用。

英文摘要：

Objective To construct and select alpha -fetoprotein （ AFP） gene transcription- regulatory dement which is active specificly in hepatoma cells. Methods The murine AFP gene enhancer Ⅰ, promoter, repressor and promoter were cloned from murine liver by PCR and recombined into eukaryotic expression vector. The recombinant pEGFP - 1 - EP contained transcription - regulatory sequence Ⅰ including enhancer Ⅰ and promoter. The recombinant pEGFP - 1 - ERP contained transcription - regulatory sequence Ⅱ including enhancer Ⅰ, repressor and promoter. The activities of two transcription -regulatory sequences in murine hepatoma cell line Hepal -6, melanoma cell line B16 were exmined by fluorescent microscope and flow cytometer apparatus by using transient transfection. Results The sequences of cloned AFP enhancer Ⅰ, repressor and promoter were identical to those reported in GeneBank. The Hepal - 6 cells transfected by pEGFP - 1 - EP and pEGFP - 1 - ERP were positive under fluorescent microscope. The Hepal -6 cells transfected by pEGFP - 1 and B16 and NIH3T3 transfected by three plasmids were negative. The rate of positive cells and mean fluorescent intensity of the Hepal - 6 cells transfected by pEGFP - 1 - EP were higher than that of the Hepal -6 cells transfected by pEGFP - 1 - ERP. Their rates of positive cells and mean fluorescent intensities were higher than that of the Hepal - 6 cells transfected by pEGFP - 1. The rates of positive cells and mean fluorescent intensities of the Hepal -6 ceils transfected by pEGFP- 1 and B16 and NIH3T3 transfected by three plasraids were lower. Conclusion The murine AFP gene regulatory sequence Ⅰ and Ⅱ are active specificly in hepatoma cell line. The activity of regulatory sequence Ⅰ is higher than regulatory sequence Ⅱ. We will select and apply regulatory sequence Ⅰ in further construction of specific gene therapy vaccine for hepatoma.