摘要
为了提高腺病毒载体用于基因治疗的靶向性,采用PCR和体外连接的方法构建了柯萨奇病毒-腺病毒受体(Coxsackievirus-AdenovirusReceptor)胞外段sCAR和表皮生长因子(Epidermalgrowthfactor)EGF融合基因,然后将此融合基因插入穿梭质粒pDC315。利用Ad-MAX腺病毒系统,将重组质粒pDC315-sCAR-EGF与腺病毒骨架质粒pBHGloxΔE13cre共同转染AD-293细胞,成功包装出一种复制缺陷型腺病毒Ad5-CMV-sCAR-EGF。经PCR鉴定该病毒含有sCAR-EGF融合基因片段,Westernblotting证实该病毒能表达sCAR-EGF融合蛋白。体外试验证实该病毒感染细胞所产生的融合蛋白能够引导携带报告基因的腺病毒Ad5-CMV-luc高水平感染肿瘤细胞,为高水平表达EGFR的肿瘤的靶向性基因治疗提供了新的手段。
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 AdMaxTM system, AD-293 cells were cotransfected with pDC315-sCAR-EGF and adenovirus genomic plasmid pBHGlox△E13cre. Through high efficiency site specific recombination, a replication-defective adenovirus AdS-CMV-sCAR-EGF was constructed. The recombinant adenovirus was analyzed by PCR and Western blotting, the results indicated that AdS-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 AdS-CMV-luc with the reporter gene in vitro, which providing a new approach to the gene therapy for tumors overexpressing EGFR.
出处
《生物工程学报》
CAS
CSCD
北大核心
2006年第5期713-719,共7页
Chinese Journal of Biotechnology
基金
国家自然科学基金杰出青年项目(No.30325043)
国家973项目(No.2004CB518800)。~~
关键词
腺病毒
表皮生长因子受体
基因治疗
柯萨奇病毒-腺病毒受体
adenovirus, epidermal growth factor receptor, gene therapy, Coxsackie virus- adenovirus receptor
