应用人延伸因子1α亚基启动子和人工转录激活因子提高外源基因在CHO细胞中的表达  

Enhancement of Foreign Gene Expression in CHO Cells by Human Elongation Factor 1α Subunit Promoter and Artificial Transcription Activator Factors

作　　者：来大志[1] 翁少洁[1] 于长明[1] 齐连权[1] 付玲[1] 于婷[1] 陈薇[1] 

机构地区：[1]北京微生物流行病研究所,北京100071

出　　处：《生物化学与生物物理进展》2004年第2期118-126,共9页Progress In Biochemistry and Biophysics

摘　　要：表达载体的设计对于提高外源基因在哺乳动物细胞中的表达量十分重要 .而表达载体中最为重要的元件是启动子 .一些常用病毒来源的启动子 ,如巨细胞病毒即早期启动子 (PCMV IE)仅在S期激活 ,应用这类启动子表达外源蛋白需要宿主细胞不停增殖 ,这对于连续持久大规模培养的宿主细胞不现实 .而人延伸因子 1α亚基启动子 (PEF 1α)不受细胞周期限制 ,且启动子强度高于PCMV IE,对于大规模外源蛋白的生产较为理想 .首先构建了基于PEF 1α启动子的哺乳动物细胞表达载体pED5 ,在增殖缓慢的CHO细胞中 ,此载体表达外源蛋白的能力是应用PCMV IE启动子表达载体的 4 1倍 .由于启动子一般仅能决定基因转录的本底水平 ,而转录激活因子 (transcriptionactivators)对基因转录的影响更大 ,所以另把两个人工转录激活结构域AH和VP2接到λ噬菌体cⅠ蛋白的C端 ,它们通过cⅠ蛋白结合于已插入到PEF 1α启动子的TATA框上游约 2 0 0bp的λ噬菌体OR2 OR1序列上 ,而被“征募”到PEF 1α启动子TATA框附近 ,从而促进基因的转录 .把上述元件均整合进一个表达载体中 ,构建了两个新型表达载体pER AH和pER VP2 .pER AH表达外源蛋白的能力比不含转录激活因子的表达载体略高 ,而pER VP2表达外源蛋白的能力则比不含转录激活因子的表达载体高Expression vectors are critical to high efficiency expression of foreign proteins. Thus a vector containing human elongation factor 1α subunit promoter (P EF 1α ) for transcription of genes of interests, and mouse dihydrofolate reductase ( dhfr ) gene under control of SV40 promoter (P SV40 ) for clonal selection and amplification was first constructed . The vector was named pED5. The expression efficiency difference between pED5 and pCdhfr1, a vector utilizing CMV enhancer/promoter (P CMV IE ) for foreign protein production, was analyzed using human interferon β (IFN β) gene and human secreted alkaline phosphatase (SEAP) gene as reporters. When analyzed in transient expression, pED5 showed a little more protein produciton than pCdhfr1. However, in continuous expression, when serum concentration was lessened to slow down cell proliferation, pED5 expressed 3 1 times more reporter proteins than pCdhfr1, which implied that P EF 1α was less affected by cell cycle status in contrast to P CMV IE , making pED5 a good expression vector for foreign protein production.To further enhance protein productivity of expression vectors, two artificial transcription activating domains, AH and VP2, were linked to cI repressor protein of phage λ through a soft linker, respectively, and thus two artificial transcription activators were created. The O R2 O R1 sequence of phage λ was inserted into P EF 1α about 200 bp before TATA box. The artificial transcription activators can bind to O R2 O R1 sequence, and are thus conscripted near to the TATA box of P CMV IE , activating gene transcription with artificial activating domain AH or VP2. All the components mentioned above were integrated into pED5, producing two vectors: pER AH and pER VP2. The two vectors were tested for their efficiency of expressing IFN β and SEAP reporter genes in comparison with pERFRT, a vector similar to pER AH and pER VP2 except for lacking of artificial transcription activators. To abolish transcripti

关 键 词：人工转录激活因子 人延伸因子1α亚基启动子 基因表达 cI蛋白 CHO细胞 

分 类 号：Q786[生物学—分子生物学]