摘要
目的:用AdEasy腺病毒载体系统构建CTLA4Ig重组腺病毒,以感染树突状细胞使其递呈抗原至T淋巴细胞功能受阻.方法:双酶切、凝胶回收方法从质粒pCDM8-CT-LA4Ig提取目的基因CTLA4Ig,构建成腺病毒穿梭质粒pAdTrack-CTLA4Ig;经酶切线性化后的pAdTrack-CTLA4Ig与AdEasy-1进行同源重组,经酶切线性化转染入HEK293细胞进行包装.结果:卡那霉素抗性筛选和PacⅠ酶切确证重组腺病毒质粒pAd-CTLA4Ig,PacⅠ酶切产生30kb和4.5kb2个片断为同源重组.重组腺病毒质粒经293细胞包装后可观察到绿色荧光,收获病毒并制备出高滴度重组腺病毒(病毒滴度为1.6×109).结论:成功构建了携带CTLA4Ig基因的重组腺病毒载体,为利用CTLA4Ig基因转染树突状细胞的基因治疗奠定了基础.
AIM： To construct recombinant adenovirus vector carrying the human cytotoxic lymphocyte antigen 4 immunoglobulin （CTLA4Ig） gene and amplify the adenovirus vector in HEK293 cells, and then to infect dendritic cells to block the submission of antigen from dendritic cells to T lymphocytes. METHODS： CTLA4Ig gene with the suitable enzyme site was double-digested with restrictive endonucleases HindⅢ and XbaI from pCDM8-CTLA4Ig vector, then subcloned into the shuttle plasmid pAdTrackCMV after digested by the same enzyme. The recombinant plasmid pAdTrack-CTLA4Ig linearized by PmeⅠ, was chemically transfected into E. coli BJ5183 cells that had been chemically transfected with adenovirus backbone plasmid pAdEasy-1. Finally, the linearized recombinant plasmid was transfected into HEK293 cells. RESULTS： Recombinants were selected by kanamycin resistence and confirmed by PacI. The restrictive endonuclease analysis confirmed that correct recombinant adenovirus plasmid was constructed （30 kb and 4.5 kb fragments were dotained after PacI digestion ）. Twenty-four hours after transfection, the fluorescence was observed in 293 cells, and viral tite was checked by GFP （ 1.6 ×10^9 ）. CONCLUSION： The recombinant adenoviral vector containing CTLA4Ig gene has been successfully constructed, which offers a basis for the gene therapy of CTLA4Ig infecting the dentritic cells.
出处
《第四军医大学学报》
北大核心
2007年第14期1279-1282,共4页
Journal of the Fourth Military Medical University
基金
国家自然科学基金(30400408)
