人甲胎蛋白基因转录调控元件的改造  

Reconstruction of Transcriptional Regulatory Elements of Human α-fetoprotein Gene

作　　者：马强中[1] 吴易元[1] 雷薇[1] 遇珑[1] 王洪平[1] 张友会[1] 

机构地区：[1]中国医学科学院中国协和医科大学肿瘤研究所,北京100021

出　　处：《中国医学科学院学报》1998年第2期114-120,共7页Acta Academiae Medicinae Sinicae

基　　金：中国医学科学院中国协和医科大学重点科研基金

摘　　要：目的为构建高效利用甲胎蛋白(AFP)基因转录调控元件(TREs),驱动治疗基因在肝癌细胞中特异性表达的基因治疗载体,对天然人AFP TREs进行改建和鉴定。方法采用亚克隆技术,删除了天然人AFP TREs中含有沉默子活性的-0.87～-3.06 kb区域,将具有增强子活性的-3.06～-5.1 kb片段和调控AFP基因表达的启动子"核心"区域+29～-870 bp片段连接,获得经人工改造后的基因转录调控元件,命名为AFP(e/p)TREs。用报告基因氯霉素乙酰转移酶(CAT)表达法测定TREs的活性和特异性。结果 AFP(e/p)TREs在人肝癌细胞FepG2中的活性是天然人AFP TREs的1.8倍,并只能使CAT基因在AFP阳性的HepG2细胞中表达而不在喉癌Hep2细胞中表达。结论改建后的AFP(e/p)TREs较天然的人AFP TREs长度缩短,具有更高的活性并仍具有肝癌细胞特异性,适合用于肝癌特异性表达基因治疗载体的构建。To construct vectors that express therapeutic genes specifically in hepatocellular carcinoma, the TREs (Tissue-specific transcriptional regulatory elements) of natural human α-fetoprotein (AFP) was reconstructed. Methods Using subcloning techniques, the-870 bp～—3.06 kb fragment of the natural human AFP TREs was deleted to construct an artificial AFP(e/p) TREs which contains the 'core' promoter and the—3.06 kb～—5.1 kb fragment of natural human AFP TREs that contains enhancer activity. Results By using chloramphenicol acetyltransferase (CAT) assay, the artificial AFP (e/p) TREs showed an activity 1.8 fold as high as that of the natural AFP TREs in an AFP positive human hepatocarcinoma cell line HepG2, and it also remained to be tissue-specific since there was no activity was detected in an AFP negative human larynxcarcinoma cell line Hep2. Conclusions These results indicate that the artificial AFP(e/p) TREs we have constructed has higher activity than the natural AFP TREs and it also remains its hepatocarcinoma specificity. The reconstructed AFP(e/p) TREs is suitable for constructing of gene therapy vector with hepatocarcinoma expression specificity.

关 键 词：甲胎蛋白 基因转录调控 基因疗法 载体 肝肿瘤 

分 类 号：R735.705[医药卫生—肿瘤]