pDC316-hIL-12-IRES-CKb双顺反子重组腺病毒载体的构建及其在肝细胞中的表达

Construction of a recombinant adenovirus carrying IL-12-IRES-CK gene and its expression in hepatocyte

目的:构建含肌酸激酶(CK)基因和人白细胞介素12(hIL-12)基因的双顺反子重组腺病毒载体pDC316-hIL-12-IRES-CKb,并包装成重组腺病毒,检测其在肝细胞中蛋白表达和肝脏中磷酸肌酸的水平。方法:将PCR扩增的产物CK片段和hIL-12片段顺次插入双顺反子腺病毒载体(IES)中,经同源重组、包装和扩增获得重组腺病毒子。再用该病毒感染体外培养的兔肝细胞,经Western blotting检测到CK蛋白的表达和ELISA检测培养液中hIL-12水平。结果:体外实验在感染肝细胞后检测到CK和hIL-12蛋白的表达;同时体内实验应用核磁共振波谱(MRS)的方法能检测到肝脏特异性CK产物磷酸肌酸的水平。结论:携带CK和hIL-12基因的双顺反子重组腺病毒能使CK基因异源性地表达在肝脏并能用非侵袭性的MRS技术检测其表达。此载体的构建为进一步研究CK基因作为一个影像报告基因动态监测肝脏治疗基因hIL-12的表达奠定基础。

AIM： To construct a bicistronic recombinant adenovirus carrying creatine kinase （CK） and human interleukin 12 （hIL-12） gene, and then detect the expression of both genes in vitro and metabolic product of CK in vivo. METHODS： Two PCR products, CK and hIL- 12 genes linked by IRES, were inserted into adenoviral vector. Adenovirus particles carrying CK-IRES-IL- 12 gene was generated through homologous recombination, packaging and propagation. Rabbit hepatocytes were isolated and transfected with adenovirus particles. CK and IL-12 gene expression was confirmed by Western blotting and ELISA, respectively.RESULTS： The expression of CK and human IL-12 in hepatocyte were confirmed in vitro. Metabolic product of CK, phosphocreatine （PCr）, could be detected in liver by non-invasive phosphorus-31 magnetic resonance spectroscopy （31P MRS） in vivo.CONCLUSION： In vivo detection of ectopic expression of CK gene in liver can be achieved by non-invasive 31P MRS through adenovirus transduction. The bicistronic recombinant adenovirus Ad5-hIL-12-IRES-CKb carrying CK and hIL- 12 provides a useful tool for the further study of using a reporter gene expression （CK） dynamically, non-invasively monitor a therapeutic gene expression （IL-12） in liver.