腺病毒介导重组人单链IL-27融合基因在肝癌细胞的表达及活性鉴定

Adenovirus mediated expression of recombinant human single chain interleukin-27(rhscIL-27) fusion gene in hepatoma cells

目的:利用腺病毒表达系统在肝癌细胞表达有生物活性的单链重组人白细胞介素-27(rhscIL-27)并检测其生物学活性。方法:将rhscIL-27基因片段从pcDNA3.1载体克隆到腺病毒载体pAdTrack-CMV,然后在细菌内与pAdEasy同源重组构建AdIL-27腺病毒载体并转染293细胞包装、扩增腺病毒颗粒。将重组腺病毒感染肝癌细胞株HepG2,荧光显微镜、RT-PCR及ELISA等方法进行检测蛋白表达情况,IFN-γ诱生实验检测rhscIL-27的生物学活性。结果:成功构建AdIL-27腺病毒载体,经293细胞包装扩增后感染HepG2细胞观察到有GFP表达,RT-PCR及ELISA检测到细胞有rhscIL-27表达,IFN-γ诱生实验证实表达的rhscIL-27具有诱导产生IFN-γ的生物学活性。结论:利用腺病毒表达系统成功在肝癌细胞表达有生物活性的重组人单链IL-27(rhscIL-27),并具有诱导产生IFN-γ的生物学活性,为研究IL-27的生物学功能及其对肝癌的基因治疗奠定了基础。

AIM：To explore the adenovirus mediated expression of recombinant human single chain interleukin-27（rhscIL-27） fusion gene in hepatoma cells. METHODS： The rhscIL-27 fusion gene was subcloned into the shuttle plasmid pAdTrack-CMV and then clone the homologous recombinant adenovirus genomic plasmid pAdEasy in bacteria. The identified recombinant plasmid AdIL-27 was tranfected into 293 cells, and then the adenovirus did the package and amplification. The HepG2 cells were infected with AdIL-27 and the target gene expression was determined by RT-PCR and ELISA. The biological activity of rhscIL-27 was detected by IFN-γ inducing assay. RESULTS： Restriction endonuclease and gene sequencing confirmed that the recombinant adenovirus vector of rhscIL-27 fusion gene was successfully constructed. The expression of rhscIL-27 fusion gene was observed at 48 h after the transfection of the HepG2 cells with AdIL-27. The IFN-γ inducing assay showed that the rhscIL-27 protein has the ability inducing IFN-γ secretion. CONCLUSION： By using adenovirus expression system, rhscIL-27 fusion gene with biological activity is expressed successfully in hepatoma cells. This experiment laid a foundation for gene therapy of hepatoma with IL-27.