表达Apoptin基因抗肿瘤双特异性重组腺病毒的构建及鉴定

Construction and identification of the dual specific anti-tumor recombinant adenovirus expressing apoptin

为研发一种安全、特异、高效的抗肿瘤基因治疗候选药物,本试验基于人端粒酶启动子(hTERTp)、人5型腺病毒复制必需基因(E1a)和特异性抑癌基因(Apoption),利用RAPAd.Ⅰ腺病毒载体系统,构建了一种具有肿瘤特异性杀伤和特异性复制能力的双特异性抗肿瘤重组腺病毒Ad-VT。并利用人肝癌细胞(HepG-2)、人肺癌细胞(A549)、人宫颈癌细胞(Hela)、人胚胎肺细胞(WI-38)和非洲绿猿肾细胞(Vero)对hTERTp的肿瘤特异性进行鉴定。结果显示,hTERTp可在HepG-2、A549和Hela等肿瘤细胞内特异性启动外源基因的表达,而在WI-38、Vero等正常细胞内不能启动相关基因表达。用RT-PCR及Western-blot等方法对Apoptin基因和E1a基因的转录和表达进行了鉴定。结果表明,Ad-VT能够有效表达Apoptin和E1a等外源基因。

To develop a safe, specific and effective succedaneous praeparatum in cancer gene therapy, a dual specific anti-tumoral recombinant adenovirus, Ad-VT, which has both tumor specific restrain and tumor specific replication a- bilities,was constructed by using RAPAd. i system basing on human telomerase reverse transcriptase core promot- er （hTERTp）, replicate indispensable gene El a and specific anti-tumor gene apoptin. The specificity of hTERTp was identified in human hepatoma carcinoma （HepG-2） cells,human lung carcinoma （A549） cells, human cervical carci- noma （Hela） cells,human embryonic lung （WI-38） cells and african green monkey kidney （Vero） cells. The expres- sion of apoptin and Ela of Ad-VT was identified by RT-PCR and Western-blot. The results showed that hTERTp had a promoter acitivity specifically in HepG-2, A549 and Hela cells, whereas not in WI 38 and Vero, and the foreign genes of Ad-VT were expressed effectively in target ceils.