XAF1基因重组腺病毒载体的构建及其在人肝癌细胞体内外的表达

Construction of XAF1 recombinant adenovirus vector and its expression in human hepatocellular carcinoma cell line both in vitro and in vivo

目的利用携带35型腺病毒纤毛的嵌合型5型腺病毒载体系统Ad5/F35构建XAF1基因重组腺病毒,体内外感染人肝癌细胞SMMC7721并使XAF1基因有效表达。方法将真核表达质粒pcDNA3.1-XAF1和穿梭质粒pDC316用BamHⅠ和EcoRⅠ双酶切、筛选、测序获得重组穿梭质粒pDC316-XAF1。将测序正确的pDC316-XAF1和骨架质粒pBHG-fiber5/F35用Lipofectamine2000共转染HEK293细胞,进行细胞内同源重组,得到重组腺病毒Ad5/F35-XAF1。予终点稀释法测定重组腺病毒的感染滴度。用同样的方法得到携带增强型绿色荧光蛋白(EGFP)的报告病毒Ad5/F35-EGFP。建立人肝癌细胞株SMMC7721裸鼠移植瘤模型,将Ad5/F35-XAF1和Ad5/F35-EGFP重组腺病毒分别感染人肝癌细胞株SMMC7721和瘤内注射;荧光显微镜观察EGFP在细胞和移植瘤冰冻切片中的表达;RT-PCR和Westrenblot法检测XAF1的mRNA和蛋白在细胞和移植瘤组织的表达。结果重组腺病毒Ad5/F35-EGFP感染肝癌细胞和瘤内注射后,予荧光显微镜均可见细胞和冰冻切片中呈现绿色荧光;Ad5/F35-XAF1感染肝癌细胞和瘤内注射后,XAF1mRNA和蛋白表达均显著高于对照组和报告病毒组。结论成功构建重组腺病毒Ad5/F35-XAF1和Ad5/F35-EGFP。该腺病毒载体可携带目的基因在人肝癌细胞株SMMC7721体内和体外进行有效表达。

Objective To construct the recombinant adenovirus vector containing XAF1 gene by Ad5/F35, a chimeric type 5 adenovirus vector system with type 35 adenovirus fiber. Human hepatocellular carcinoma cell line SMMC7721 was infected by this recombinant adenovirus vector and the XAF1 gene was expressed effectively both in vitro and in vivo. Methods The eukaryotic expressive plasmid pcDNA3. 1-XAF1 and the shuttled plasmid pDC316 were both digested by restriction enzymes with BamH I and EcoR I. The recombinant shuttle plasmid pDC316-XAF1 was generated by selection and sequence. Thereafter, pDC316-XAF1 was co-transfected with backbone pBHG-fiber5/F35 plasmid into HEK293 cells to establish the recombinant adenovirus Ad5/F35-XAF1 by homologous recombination. The infective titer of recombinant adenovirus was tested by terminal dilution. The report adenovirus Ad5/F35-EGFP was constructed by the same method as the report adenovirus. The model of nude mice beared xenograft by human hepatocellular carcinoma cell line SMMC7721 was constructed by subcutaneously injected into the cells in the right armpit of nude mice. The recombinant adenovirus Ad5/F35-EGFP and Ad5/F35-XAFI were infected into SMMC7721 cell line and injected into xenografted tumors respectively. Then the expression of EFGP was observed by fluorescence microscope. The mRNA and protein expression of XAF1 were testified by RT-PCR and Western blot both in cells and xenografted tissues, respectively. Results The green fluorescence of EGFP was observed both in cells and in frozen slides of xenografted tumor tissues after Ad5/F35-EGFP infected the cells and tumors. The expression of XAF1 was higher in Ad5/F35-XAF1 treated group both in cells and tumor tissues than in control and Ad5/F35-EGFP group. Conclusions The recombinant adenovirus Ad5/F35-EGFP and Ad5/F35-XAF1 were established successfully. The target gene can be contained by recombinant adenovirus vector and expressed effectively in human hepatocellular carcinoma cell line both in vitro and in vivo.