重组腺病毒介导的基因转移系统的建立以及在离体及活体中表达

Establishment of the recombinant adenovirus-mediated gene transfer system and expression of hf Ⅸ in vivo and in vitro

首先建立了重组腺病毒载体介导的基因转移系统：将质粒ＰＡｄＣＭＶｌａｃＺ用磷酸钙沉淀法转移至２９３腺病毒包装细胞中，经挑克隆、病毒扩增、纯化、浓度和滴度测定，制备了纯化的病毒颗粒ＡｄＣＭＶｌａｃＺ；以此感染离体细胞，ｌａｃＺ基因转移效率可达９０％～９５％；小鼠被直接活体注射重组的腺病毒ＡｄＣＭＶｌａｃＺ，ｌａｃＺ基因能够在多种组织中表达．其次，运用重组腺病毒介导的基因转移系统，进行了人凝血因子ⅨｃＤＮＡ（ｈＦⅨｃＤＮＡ）的基因构建、转移和表达研究：构建合ｈＦⅨｃＤＮＡ和腺病毒片段的质粒（ＰＡｄＣＭＶｈＦⅨ），与质粒ＰＪＭ１７共转染２９３细胞，制备含ｈＦⅨ的重组腺病毒（ＡｄＣＭＶｈＦⅨ），获得在离体细胞和活体组织中高效表达．

n this paper,the recombinant adenovirus-mediated gene transfer system was established. In the system,the efficiency for lacZ gene transfer in vitro was 90%￣95 %and lacZ gene was also expressed in multiple tissues in mice by injection of the recombinant adenovirus AdCMVlacZ. Moreover,by the use of this transfer system,the human coagulation factor Ⅸ (hFⅨ ) could be expressed in vitro and in vivo with high efficiency.