可溶性肿瘤坏死因子相关凋亡诱导配体重组腺病毒载体的构建及其在肝癌细胞株中的表达

Construction of Recombinant Adenovirus Containing Soluble Tumor Necrosis Factor-related Apoptosis-inducing Ligand Gene and its Expression in Liver Cancer Cell Line

背景:肿瘤坏死因子相关凋亡诱导配体(TRAIL)能诱导30余种人类肿瘤细胞凋亡,在抗肿瘤治疗中具有广阔应用前景。目的:构建携带可溶性TRAIL基因的复制缺陷型重组腺病毒载体,鉴定其在肝癌细胞株中的表达。方法:以重叠延伸聚合酶链反应(PCR)扩增白细胞介素(IL)-2信号肽和TRAIL膜外区融合片段,克隆于腺病毒穿梭质粒pAdTrack-CMV,转化含复制缺陷型腺病毒骨架质粒pAdEasy-1的大肠杆菌pAdBJ5183,经细菌内同源重组产生重组腺病毒Ad-IL-2-TRAIL,以脂质体法转染HEK293细胞包装病毒,感染肝癌细胞株HepG2,荧光显微镜下观察增强型绿色荧光蛋白(EGFP)的表达。结果:成功构建了携带可溶性TRAIL基因的复制缺陷型重组腺病毒载体。病毒感染HepG2细胞48h后,超过95%的细胞中出现绿色荧光。结论:所构建的复制缺陷型重组腺病毒Ad-IL-2-TRAIL可在肝癌细胞中高效表达可溶性TRAIL,为进一步行肿瘤TRAIL基因治疗提供了实验依据。

Background： Tumor necrosis factor-related apoptosis-inducing ligand （TRAIL） can induce apoptosis in more than 30 kinds of human cancer cells, and has great potential in the treatment of cancers. Aims： To construct a replicationdefective recombinant adenovirus containing soluble TRAIL gene, and to identify its expression in liver cancer cell line. Methods： The fusion gene of interleukin （IL）-2 signal peptide and soluble TRAIL was amplified by overlap extension polymerase chain reaction （PCR）, and the product was cloned into adenoviral shuttle plasmid pAdTrack-CMV. Then the recombinant shuttle plasmid was transformed into Escherichia coli pAdBJ5183 containing skeleton plasmid pAdEasy-1 of replication-defective adenovirus, the positive clone of homologous recombination was selected. The recombinant Ad-IL-2- TRAIL was transfected into HEK293 cells by LipofectamineTM 2000 to package adenovirus particles, and then infected the liver cancer cell line HepG2. The enhanced green fluorescent protein （EGFP） expressed by HepG2 cells was examined by fluorescent microscope. Results： Replication-defective recombinant adenovirus containing soluble TRAIL gene was successfully constructed. More than 95% HepG2 cells expressed EGFP 48 hours after infection with adenovirus particles. Conclusions： Soluble TRAIL can be highly expressed in liver cancer cells by infection with replication-defective recombinant adenovirus Ad-IL-2-TRAIL, which provides the possibility of TRAIL gene therapy for cancers.