鸡贫血病毒VP 3基因的克隆及其体外凋亡诱导效应的研究

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pcDNA3上,构建了重组体pcDNA-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LipofectAMINETM介导的基因转染,将pc-DNA-vp3、pcDNA3分别转入肝癌细胞系HepG2和二倍体肝细胞系L-02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.

Cloning of Chicken Anemia Virus vp3 Gene and Study on the Apoptosis Inductive Effect of vp3 Gene in vitro

Using PCR technique, the vp3 gene of chicken anemia virus (CAV) was cloned into the eukaryotic expression vector pcDNA3 to construct a recombinant pcdNA vp3. Restriction enzyme and sequencing analysis revealed that CAV vp3 gene was correctly inserted into the blank vector pcDNA3. Total mRNA was extracted from liver carcinoma cell lines HepG2 and diploid cell line L 02 after LipofectAMINE TM mediated transfection in vitro with pcDNA vp3. After that, RT PCR was performed. The results of RT PCR suggested that vp3 gene was expressed in these two cell lines. At the same time, using in situ apoptotic detection assay, TUNEL Kits, the apoptotic cells were found in pcDNA vp3 transfected HepG2, but not in mock transfected cell lines. vp3 could induce cell death by apoptosis. vp3 could only induce apoptosis in cancer cell lines, but not in diploid cell lines. All the results indicated that CAV vp3 gene, a potential therapeutic agents, would be used widely in treating cancers in future