应用AdMax系统构建携带HCV核心基因Core的重组腺病毒载体

Construction of Recombinant Adenovirus Vector with HCV Core Gene by AdMax System

目的应用AdMax系统构建携带丙型肝炎病毒(HCV)核心基因Core的重组腺病毒载体,为进一步研究防止HCV感染的基因疫苗和基因治疗奠定基础。方法RT-PCR法从丙型肝炎患者血清中扩增HCVCore基因,连接到腺病毒穿梭质粒的多克隆位点上,构建重组穿梭质粒pDC315-Core,在脂质体介导下与腺病毒辅助大质粒pBHGlox(delta)E1,3Cre共转染HEK293细胞,包装产生复制缺陷型重组腺病毒pAd-Core,经HEK293细胞扩增后,离子交换柱层析法纯化病毒,测定病毒颗粒数及滴度,并计算比活性及单细胞产量。结果重组腺病毒pAd-Core感染的HEK293细胞经PCR检测,可扩增出HCV核心基因Core片段,经测序证明,其插入序列与设计的HCV Core基因序列一致。扩增纯化后,重组腺病毒的比活性达0.034IU/VP,单细胞产量可达2.63×104VP/细胞。结论已成功构建重组腺病毒载体pAd-Core,为Core基因在腺病毒介导下的基因免疫和基因治疗的研究奠定了基础。

Objective To construct the recombinant adenovirus vector carrying human hepatitis C virus （HCV） Core gene by AdMax system and lay a foundation of further development of gene vaccine against HCV infection as well as gene therapy of HC. Methods HCV Core gene was amplified from the sera of patients with HC by RT-PCR and inserted to the polyclonal site of adenovirus shuttle plasmid pDC315. HEK293 cells were co-transfeeted with the constructed recombinant shuttle plasmid pDC315-Core and large adenovirus helper plasmid pBHGlox （delta） E 1, 3Cre in mediation of liposome. The obtained replication-defective recombinant adenovirus pAd-Core was propagated in HEK293 cells, purified by ion exchange chromatography, counted for virus particles and determined for titer, based on which specific activity and the yield of virus particles in a single cell were calculated. Results HCV Core gene fragment was amplified by PCR from the HEK293 cells transfected with pAd-Core. Sequencing result proved that the inserted sequence was identical to the designed HCV core gene sequence. After propagation and purification, the specific activity and yield of recombinant adenovirus pAd-Core were 0. 034 IU/VP and 2. 63 × 10^4 virus particles per cell respectively. Conclusion Recombinant adenovirus vector pAd-Core was successfully constructed, which provided a basis for application of HCV Core gene to adenovirus-mediated gene immunization and gene therapy.