CAJ | 학술논문

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-CeuⅠ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pAdeno-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.
목적:구건함인AFP기인적선병독재체,체외전염수돌상세포,제비수돌상세포간암류묘.방법: 장AFP기인아극륭도pIND 재체화Shuttle2재체중,구건천사재체Shuttle2-AFP.용PI-Sce Ⅰ화I-CeuⅠ쌍매절후장소획AFP기인편단재여선성화적선병독재체pAdeno-X련접,구성pAdeno-AFP중조선병독재체.기후,용중조선병독재체전염HEK293세포,포장선병독표체재체.통과매절、PCR대선병독재체진행감정.포장호적중조병독재체pAdeno-AFP체외감염수돌상세포제비수돌상세포간암류묘후,FACS분석pAdeno-AFP/DC표면분자적표체,매련면역흡부시험법( ELISA) 검측AFP수평.결과: 매절、PCR감정증실,천사질립삽입편단위AFP기인.포장적선병독재체구유량호적감염성,가이재293세포중형성병독과립,선병독재체내휴대AFP기인감염수돌상세포,pAdeno-AFP/DC능고수평적표체CD1a,CD11c,CD80,CD86이급HLA-DR,병분비교고수평적AFP.결론: 구건성공적함AFP선병독재체가이재수돌상세포중표체AFP,위DC류묘적진일보연구전정료기출.
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 shuttle2 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 adenovirus 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 CD1a,CD11c,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.