目的：构建编码融合基因NT4-p53(N15)-Ant的重组腺病毒表达载体，为进一步基因治疗的实验研究奠定基础。方法：利用PCR体系延伸互为模板的引物，通过T载体克隆法获得p53(N15)-Ant基因克隆，酶切后连入pBV220/NT4质粒，再将融合基因NT4-p53(N15)-Ant亚克隆至腺病毒的穿梭质粒内，与辅助质粒PJM17共同转染HEK-293细胞，通过同源重组获得重组腺病毒Ad.NT4-p53(N15)-Ant，收集病毒上清，大量扩增并测定其滴度，用RT-PCR检测目的基因在293细胞的表达情况。MTT比色法观察重组病毒对HepG2细胞存活率的影响。结果：克隆出p53(N15)-Ant基因，经酶切及测序证实结果正确；得到高滴度的（1×1011pfu/ml）重组腺病毒表达载体；反转录聚合酶链反应证实感染Ad.NT4-p53(N15)-Ant的293细胞中有目的基因的表达。Ad.NT4-p53(N15)-Ant对HepG2细胞有强烈的杀伤作用,与Ad.GFP比较，NT4 p53(N15)Ant重组腺病毒处理组HepG2细胞的存活率明显降低。结论：通过分子克隆体外重组技术成功制备了NT4-p53(N15)-Ant复制缺陷型重组腺病毒，为下一步的肿瘤基因治疗奠定了基础。

Objective: To construct a recombinant adenovirus vector harboring fusion gene NT4-p53(N15)-Ant , laying a foundation for gene therapy research of malignant tumors. Methods: The p53(N15)-Ant gene was obtained by T-vector method and was inserted in pBV220/NT4 vector after digested with restriction enzyme. The fusion gene of NT4-p53(N15)-Ant was subcloned into the shuttle plasmid of adenovirus; the products were cotransfered into HEK-293 cell line with helper plasmid PJM17. The recombinant adenovirus was produced by homologous recombination of above 2 plasmids in HEK-293 cells and its titer was measured by plaque-forming. The expression of Ad.NT4p53Ant in transfected 293 cells was confirmed by reverse transcription polymerase chain reaction (RT-PCR) procedure. The effect of Ad.NT4p53Ant on HepG2 cell line was measured by a colorimetric 3-(4, 5-dimethyl-2-thiazolyl)-2, 5-diphenyl-2H-tetrazolium bromide (MTT) assay. Results:The -p53(N15)-Ant gene was confirmed by restriction enzyme digestion and DNA sequencing. High titer of recombinant adenovirus was obtained by homologous recombination in HEK-293 cells (1×1011pfu/ml). The expression of NT4-p53(N15)-Ant gene in 293 cells was confirmed by RT-PCR. Ad. NT4p53Ant had strong killing effect on HepG2 cells. Compared with Ad.GFP, Ad.NT4p53Ant significantly decreased the survival rate of HepG2 cells. Conclusion: The recombinant adenovirus vector encoding gene NT4-p53(N15)-Ant has been successfully constructed in this experiment by molecular cloning and in vitro recombination techniques，laying a foundation for further research of gene therapy of cancer.

国家自然科学基金资助项目（30471942），陕西省科技攻关项目（2004k11 G3）