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用 PCR方法扩增了鸡贫血病毒标准株的 vp3基因 ,并将其克隆于真核表达载体 pc DNA3上 ,构建了重组体pc DNA- vp3。经酶切鉴定及测序分析表明 ,该片段和预期相符。在体外利用 L ipofect AMINETM介导的基因转染 ,将 pc D-NA- vp3、 pc DNA3分别转入肝癌细胞系 Hep G2和二倍体肝细胞系 L- 0 2中 ,转染后的 RT- PCR结果证实 vp3基因在细胞中得到了表达。同时利用筛选稳定表达细胞株的技术和原位细胞凋亡检测法 ,证明了鸡贫血病毒是以凋亡的方式诱导细胞死亡 ,并且只诱导癌细胞的凋亡 ,而不诱导正常或二倍体细胞死亡。表明鸡贫血病毒 vp3基因很可能成为一种极具潜力的抗肿瘤生物制剂
The vp3 gene of chicken anemia virus standard strain was amplified by PCR and cloned into the eukaryotic expression vector pcDNA3 to construct the recombinant pcDNA-vp3. The enzyme digestion and sequencing analysis showed that the fragment was in line with the expectation. Pc D-NA-vp3 and pcDNA3 were transfected into Hep G2 cell line and L-0 2 diploid hepatocyte line respectively by L ipofect AMINETM mediated gene transfection in vitro. RT- PCR results confirmed that the vp3 gene was expressed in the cells. At the same time, by using the technology of screening stably expressing cell lines and in situ cell apoptosis assay, it was demonstrated that chicken anemia virus induces cell death by apoptosis and induces apoptosis of cancer cells without inducing normal or diploid Cell death. Show that the chicken anemia virus vp3 gene is likely to become a highly potential anti-tumor biological agents