以AdMax载体系统构建携带HCV NS5B基因的重组腺病毒表达载体

目的构建表达丙型肝炎病毒(HCV)非结构基因NS5B的重组腺病毒表达载体,并对其相关指标进行鉴定,为进一步研究防止丙型肝炎感染的基因免疫和基因治疗奠定实验基础。方法应用基因工程技术将HCVNS5B基因定向克隆至穿梭质粒pDC316上,利用脂质体介导的方法将AdMax腺病毒包装系统的骨架质粒pBHGloxΔE1、3Cre和穿梭质粒pDC316-NS5B共转染293细胞,进行同源重组,产生重组腺病毒pAd-NS5B并进行鉴定、反复感染293细胞进行扩增,扩增后测定重组病毒滴度。结果重组病毒颗粒pAd-NS5B经双引物PCR、凝胶电泳证明插入片段与HCV非结构基因NS5B片段大小相符;经测序证明其插入序列与设计HCV NS5B基因序列完全一致,扩增后重组病毒滴度达到2.3×1013IU/L。结论成功构建了表达HCV NS5B基因的重组腺病毒载体。

Construction of Recombinant Adenovirus Vector with HCV Gene NS5B by AdMax system

Objective To construct the recombinant adenovirus vector of HCV gene NS5B for the genetic immunization and gene therapy against HCV infection.Methods The fragments of HCV NS5B gene were cloned into the shuttle plasmid pDC316 to construct the recombinant shuttle plasmid pDC316-NS5B.The recombinant shuttle plasmid,adenovirus genomic plasmid pBHGloxΔE1 and 3Cre were transfected into HEK293 cells to construct the recombinant adenovirus pAd-NS5B.The PCR and sequence analysis were used to detect the target gene fragments.After the infection,the viral titer in cell 293 was examined.Results The titer was 2.3×1013 IU/L.The recombinant adenovirus was verified by PCR and sequencing.Conclusion A recombinant adenovirus vector of human HCV NS5B was successfully constructed.