腺病毒介导的HBsAg基因修饰树突状细胞瘤苗的制备

Generation of adenovirus-mediated HBsAg gene modified dendritic cells

目的:构建含人HBsAg基因的腺病毒载体,体外转染树突状细胞,制备树突状细胞肝癌瘤苗。方法:将HBsAg基因亚克隆到pIND载体和Shuttle2载体中,构建穿梭载体Shuttle2-S。用PI-SceⅠ和I-CeuⅠ双酶切后将所获HBsAg基因片段再与线性化的腺病毒载体pAdeno-X连接,构成pAdeno-S重组腺病毒载体。其后,用重组腺病毒载体转染HEK293细胞,包装腺病毒表达载体。通过酶切、PCR对腺病毒载体进行鉴定。包装好的重组病毒载体pAdeno-S体外感染树突状细胞制备树突状细胞肝癌瘤苗后Westernblot法检测其表达,流式细胞仪检测其能否诱导树突状细胞凋亡。结果:酶切、PCR鉴定证实,穿梭质粒插入片段为HBsAg基因。包装的腺病毒载体具有良好的感染性,可以在293细胞中形成病毒颗粒,腺病毒载体内携带HBsAg基因感染树突状细胞,经Westernblot法鉴定证实能够表达转染基因,并且不会诱导树突状细胞凋亡。结论:构建成功的含HBsAg腺病毒载体可以在树突状细胞中表达HBsAg,为DC瘤苗的进一步研究奠定了基础。

Objective：To construct recombinant adenovirus vectors containing human HBsAg genes, and infect dendritic cell. Methods- Full length HBsAg cDNAs were subcloned into pIND vector , followed by being cloned into shuttle2 vector. The HBsAg gene fragments resulted from the shuttle2 - S digested with PI - Sce and I - Ceu were linked to the linear adeno -X virus DNA. After packaged with HEK293 cells, the adenovirus expression vector was obtained. The plasmid pAdeno - S was identified by endonuclease and PCR. After dendritic ceils were infected pAdeno - S, the expressive activity of adenovirus vector was identified by Western blot. And apoptotic analysis was performed in recombinant pAdeno -S infected DCs by Flow cytometric. Results： HBsAg gene in the inserted DNA of adeno- S was confirmed by PCR, and predictive fragments proved by restriction enzyme digestion analysis were exhibited. All the above results indicated that human HBsAg gene had been connected with pAdeno - X vectors correctly. The recombinant adenovirus vector of human HBsAg gene packaged in HEK293 cells, it will be used to introduce the target gene into dendritic cell. Western blot analysis showed that HBV surface antigen gene was expressed in transfected DCs. And adeno - S infection had no appreciable effect on apoptosis of DCs. Conclusion ： The recombinant adenovirus vector of human HBsAg gene have been constructed successfully. The established HBsAg - DC vaccine may be a tool of the hepatocellular carcinoma immunotherapy, and it will be foundation of future clinical use of DC vaccine .