CHO细胞高效表达载体的优化  

Construction and evaluation of high expression vectors in mammalian cells with Chinese hamster EF1-α gene

作　　者：刘珊[1] 刘志刚[1] 张国强[1] 俞炜源[1] 

机构地区：[1]军事医学科学院生物工程研究所,北京100071

出　　处：《军事医学科学院院刊》2006年第4期301-305,共5页Bulletin of the Academy of Military Medical Sciences

基　　金：国家高技术研究发展计划("863"计划)资助项目(2001AA215351)

摘　　要：目的:研究分析中国仓鼠EF1-α基因的转录调控序列,以构建新型高效表达载体。方法:利用常规分子生物学技术,构建了一系列基于CHEF1-α基因的非翻译区调控序列元件的新型真核表达载体。以组织型纤溶酶原激活剂突变体(k2tPA)作为报告基因,利用本室已建立的CHO-dhfr--Z+定点整合表达系统比较载体表达外源基因的能力,挑选表达量高的载体。进一步添加翻译增强子(H213及V163)序列,再进行评价。结果:CHEF1-α5′UTR及启动子+3′UTR 1.65 kb、CHEF1-α5′UTR及启动子+3′UTR 2.7 kb这2种组合均可有效提高目的基因tPA的表达,表达效率与对照载体相比分别提高了65.9%和54.5%。在添加翻译增强子V163后,载体pMCEdEF53Ⅲ-163 frt-k2 tPA的表达效率为对照载体的2.65倍。结论:该载体系统在重组蛋白药物的研发方面具有良好的应用前景。Objective： To construct new mammalian expression vectors and evaluate the expression level of foreign gene with the transcription regulatory sequences from Chinese hamster EF1-α gene. Methods： A series of expression vectors with the transcription regulatory sequences from Chinese hamster EF1-α gene were constructed by using standard procedure. The expression efficiency of these vectors was evaluated by using the targeted integration system, based on the assay of k2tPA（ a mutant of tPA）activity by monitoring fibrin clearance. Furthermore, two vectors which included the translational enhancer H213 and V163 were selected to appraise the expression efficiency. Results： The recombinant expression vectors with two regulatory elements combination （ CHEF1-α5′UTR ＋ 3′UTR 1.65 kb, CHEF1-α5′UTR ＋ 3′ UTR 2.7 kb） increased the expression efficiency by 65.9% and 54.5% respectively. When the translational enhancer V163 was added, the highest expression efficiency of the vector could reach 2.65 times that of the control vector. Conclusion： The CHEF1-α vectors will be promising in the high-level protein expression study.

关 键 词：CHEF1—α基因 CHO/dhfr^-细胞 基因表达 组织型纤溶酶原激活物 

分 类 号：Q786[生物学—分子生物学]