Ren Peng-Kang, Wang Feng, Li Hui-Ming, Li Zong-Hai, Huang Qian
Central Experimental Laboratory, The First People's Hospital, Shanghai, China.
Sheng Wu Gong Cheng Xue Bao. 2006 Sep;22(5):713-9.
To improve the targeting of adenovirus vector for gene therapy, a fusion gene sCAR-EGF, in which epidermal growth factor gene was fused to the 3' end of extracellular Coxsackie virus-adenovirus receptor gene, was constructed and cloned into shuttle plasmid pDC315 to obtain a recombinant plasmid pDC315-sCAR-EGF. With the AdMax system, AD-293 cells were co-transfected with pDC315-sCAR-EGF and adenovirus genomic plasmid pBHGloxdeltaE13cre. Through high efficiency site specific recombination, a replication-defective adenovirus Ad5-CMV-sCAR-EGF was constructed. The recombinant adenovirus was analyzed by PCR and Western blotting, the results indicated that Ad5-CMV-sCAR-EGF contained the fusion gene sCAR-EGF, and the adenovirus infected cells was induced to produce and secrete the fusion protein into the supernatant. We have demonstrated that the fusion protein sCAR-EGF is helpful for elevating the infection efficiency of Ad5-CMV-luc with the reporter gene in vitro, which providing a new approach to the gene therapy for tumors overexpressing EGFR.
为提高腺病毒载体在基因治疗中的靶向性，构建了一种融合基因sCAR-EGF，即将表皮生长因子基因融合到细胞外柯萨奇病毒-腺病毒受体基因的3'末端，并将其克隆到穿梭质粒pDC315中，获得重组质粒pDC315-sCAR-EGF。利用AdMax系统，将pDC315-sCAR-EGF与腺病毒基因组质粒pBHGloxdeltaE13cre共转染AD-293细胞。通过高效位点特异性重组，构建了复制缺陷型腺病毒Ad5-CMV-sCAR-EGF。通过PCR和Western印迹分析重组腺病毒，结果表明Ad5-CMV-sCAR-EGF含有融合基因sCAR-EGF，且腺病毒感染的细胞被诱导产生并将融合蛋白分泌到上清液中。我们已证明融合蛋白sCAR-EGF有助于提高Ad5-CMV-luc与报告基因在体外的感染效率，这为过表达EGFR的肿瘤基因治疗提供了一种新方法。
