CHO/dhfr^-细胞定点整合高效表达系统的建立  

Development of Site-specific Integration System to High-level Expression Recombinant Proteins in CHO Cells

作　　者：周宏[1] 刘志刚[1] 孙志伟[1] 俞炜源[1] 

机构地区：[1]军事医学科学院生物工程研究所蛋白质工程研究室,北京100071

出　　处：《生物工程学报》2007年第4期756-762,共7页Chinese Journal of Biotechnology

基　　金：国家高技术"863"计划基金资助(No2001AA215351)~~

摘　　要：为了克服随机整合建立高表达细胞株时“位置效应”所带来的不可预知的后果,我们尝试建立基于定点整合的CHO高效表达系统。首先设计一个新的高效筛选载体pMCEscan。该载体含有报告基因(k2tPA)、扩增基因(dhfr)、重组酶识别序列(FRT)及筛选基因(neo),且neo基因的表达经过系统的弱化,确保能够对基因组中的整合位点进行大规模的高效筛选。然后利用该载体转染CHO/dhfr-细胞并进行大规模筛选以获得足够多的阳性克隆,并对阳性克隆进行系统分析,筛选出报告基因表达水平高、单拷贝且扩增效果好的克隆,此克隆被认为筛选载体整合入CHO细胞基因组中转录热点(Hotspot)区域,从而获得了能够实现外源基因在基因组中定点整合和有效表达的CHO/dhfr-细胞系。随后利用位点特异性重组系统(FLP-FRT)将外源基因定点整合到Hotspot区域,以实现外源基因在CHO细胞基因组中的定点整合及高效表达。并利用该细胞系实现了k2tPA的高表达,表达量达到17.1μg/106cell.24h。该研究致力于CHO细胞基因组中高表达位点的寻找和确认,建立基于定点整合的哺乳动物细胞高效表达系统。Stable transformants for mammalian cells from gene transfer often show extreme variability in expression of the introduced transgene. This occurs from the highly variable number of copies integrated into the genome and from position effects on gene expression due to random integration. We constructed engineered CHO strains that can be used for high-level production of foreign proteins by gene-targeting. After transfecting dihydroforate reductase （DHFR）-deficient CHO cells with a newly screening vector plasmid pMCEscan, which carrying a FRT-neo^ ＊ -1RES-k2 tPA fusion gene and a DHFR gene, we screened colonies by k2 tPA expression level. We selected 7 clones that expressed high level of k2 tPA and carried one copy of the plasmid in their chromosomes. These clones showed in high level k2tPA production without amplification. So we targeted reporter gene （k2 tPA） to test the basal expression ability of these cells clones. The clone, 8-1, showed the same effect to high base expression level. In this clone, the FRT-neo^＊ -IRES-tPA gene was integrated at a transcription-active, DHFR-mediated, gene-amplifiable locus in the chromosomes. A gene-targeting vector, carrying a FRT-fused hygromycin-resistance gene, was constructed to target desired genes in chromosomal FRT by FLP recombinase-mediatcd site-specific recombination. Using this cell-vector system, we could reproducibly obtain high producers of recombinant proteins by gene-targeting and gene amplification. Using the site-specific integration CHO/dhfr^- cell line 8-1, the expression level of k2tPA could amount to 17. 1μg/10^6cell·24h.

关 键 词：CHO细胞 定点整合 高效表达 重组蛋白 

分 类 号：Q78[生物学—分子生物学]