中文摘要：

目的构建编码融合基因NT4-p53（N15）-Ant的重组慢病毒表达载体，观察其对肝癌细胞的杀伤作用。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亚克隆至慢病毒的表达质粒内，与辅助质粒共同转染HEK-293细胞，通过同源重组，获得融合基因重组慢病毒LV-NT4-p53（N15）-Ant。收集病毒上清，大量扩增并测定其滴度，用逆转录聚合酶链反应（RT-PCR）检测目的基因在HepG2细胞中的表达情况。用LV-NT4-p53（N15）-Ant处理HepG2肝癌细胞，通过光镜、电镜、四甲基偶氮唑蓝（MTT）比色试验、乳酸脱氢酶（LDH）释放法和Annexin V—PI双染实验，研究LV-NT4-p53（N15）-Ant在体外对HepG2细胞生长的影响。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（1×10^11pfu／ml）的重组慢病毒表达载体。RT—PCR证实，感染LV．NT4-p53（N15）-Ant的HepG2细胞中有目的基因的表达。在感染后24、48、72h，LV．NT4-p53（N15）．Ant处理组的细胞存活率分别为83．4％、46．9％和33．9％，与LV．EGFP组比较，差异有统计学意义（P〈0．01）。LV．NT4-p53（N15）-Ant处理组细胞在感染48、72、96h后，LDH含量分别为682、815和979 IU／L，与LV.EGFP组比较，差异有统计学意义（P〈0．01），可能与肿瘤细胞膜的破坏有关。结论通过分子克隆体外重组技术，成功制备了NT4-p53（N15）．Ant复制缺陷型重组慢病毒。LV．NT4-p53（N15）-Ant对肝癌细胞具有杀伤能力，为今后的肿瘤基因治疗提供了新的可能性。

英文摘要：

Objective To construct a recombinant lentivirus vector containing fusion gene NT4-p53 （ NI5 ） -Ant and transfer it into HepG2 cancer cells for gene therapy. Methods The gene of p53 （ NI5 ） -Ant was obtained by T-vector method. After restriction enzyme digestion, the interest gene of p53 （ N15 ） -Ant was inserted in pBV220/NT4 vector and fusion gene of NT4-p53 （N15）-Ant was subcloned into the plasmid of lentivirus and eotransfered into HEK-293 cells with helper plasmid. The recombinant lentivirus was produced by homologous recombination of the above mentioned two plasmids in HEK-293 cells and its titer was measured by plaque-forming. The expression of LV. NT4-p53-Ant in transfected HepG2 cells was finally confirmed by reverse transcription polymerase chain reaction （RT-PCR） procedure. The effect of LV.NT4- p53 （N15）-Ant on HepG2 cells was measured by a colorimetric 3-[4, 5-dimethyl thiazol-2-yl ]-2, 5- diphenyhetrazolium bromide （MTF） assay. The inhibition effect on HepG2 cells of LV.NT4- p53 （N15）-Ant and its potential mechanism was detected by light microscopy, electron microscopy, MTY, LDH-release assay and annexin V-PI double staining. Results The gene of p53（N15）-Ant was confirmed by restriction enzyme digestion and DNA sequencing. High titer of the recombinant lentivirus was obtained by homologous recombination in HEK-293 cell lines （1×10^11pfu/ml）, and the expression of NT4-p53 （N15）-Ant gene in HepG2 cells was confirmed by RT-PCR. The viability of HepG2 cells was decreased to 83.4% , 46.9% and 33.9% ,at 24 h, 48 h and 72 h, respectively, after infection by LV.NT4-p53 （N15）-Ant. Compared with the LV. EGFP control group, there were significant differences （P 〈0.01 ）. The LDH level in HepG2 cells infected by IN.NT4-p53 （ N15 ） -Ant at 48 h, 72 h and 96 h after infection was 682 IU/L, 815 IU/L and 979 IU/L, respectively, significantly increased than that in the LV.EGFP group （P 〈0.01 ） , indicating the cell membrane destruction. Conclusion The re