含人AFP基因重组腺病毒载体的构建及其转染树突状细胞瘤苗的制备  

Construction of recombinant adenovirus vector containing AFP and generation of adenovirus-mediated AFP gene modified dendritic cells vaccine

作　　者：杨静悦[1] 曹大勇[2] 刘文超[1] 斯小明[1] 

机构地区：[1]第四军医大学西京医院肿瘤中心,细胞生物学国家重点学科,陕西西安710032 [2]第四军医大学西京医院肝胆外科,陕西西安710032

出　　处：《现代肿瘤医学》2009年第8期1419-1422,共4页Journal of Modern Oncology

基　　金：国家自然科学基金(30672391);全军医药卫生科研基金(06MA205);陕西省自然科学研究项目基金(2004C222)

摘　　要：目的:构建含人AFP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AFP基因亚克隆到pIND载体和Shuttle2载体中,构建穿梭载体Shuttle2-AFP。用PI-Sce Ⅰ和I-Ceu Ⅰ双酶切后将所获AFP基因片段再与线性化的腺病毒载体pAdeno-X连接,构成pAdeno-AFP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-AFP体外感染树突状细胞制备树突状细胞肝癌瘤苗后,FACS分析pAd-eno-AFP/DC表面分子的表达,酶联免疫吸附试验法(ELISA)检测AFP水平。结果:酶切、PCR鉴定证实,穿梭质粒插入片段为AFP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AFP基因感染树突状细胞,pAdeno-AFP/DC能高水平的表达CD1a,CD11c,CD80,CD86以及HLA-DR,并分泌较高水平的AFP。结论:构建成功的含AFP腺病毒载体可以在树突状细胞中表达AFP,为DC瘤苗的进一步研究奠定了基础。Objective：To construct recombinant adenovirus vectors containing human AFP genes, and infect dendritic cell. Methods： Full length AFP cDNAs were subcloned into pIND vector, followed by being cloned into shut- tle2 vector. The AFP gene fragments resulted from the shuttle2 - AFP 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- AFP was identified by endonuclease and PCR. After dendritic cells were infected pAdeno - AFP ,the surface molecules of pAdeno -AFP/DC were analysed by flow cytometry. AFP levels in culture supernatant of pAdeno - AFP/DC were measured by ELISA. Results： AFP gene in the inserted DNA of adeno - AFP was confirmed by PCR, and predictive fragments proved by restriction enzyme digestion analysis were exhibited. All the above results indicated that human AFP gene had been connected with pAdeno - X vectors correctly. The recombinant ade- novirus vector of human AFP gene packaged in HEK293 cells,it will be used to introduce the target gene into dendritic cell. pAdeno- AFP/DC were able to upregulate CDla, CDllc, CD80, CD86 and HLA -DR. And pAdeno- AFP/ DC could secrete high level of AFP in vitro. Conclusion： The recombinant adenovirus vector of human AFP gene have been constructed successfully. The established AFP - DC vaccine may be a tool of the hepatocellular carcinoma immunotherapy, and it will be the foundation of future clinical use of DC vaccine.

关 键 词：树突状细胞 腺病毒载体 AFP 转染 

分 类 号：R735.7[医药卫生—肿瘤]