IIn-UK融合基因在CHO细胞中的高表达研究  

High expression of IIn-UK fusion gene in CHO cell using new mammalian expression vector

作　　者：刘志刚[1] 林建波[1] 张国强[1] 安怀杰[1] 徐桂清[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》2004年第3期209-211,224,共4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( 2 0 0 1AA2 15 3 5 1)

摘　　要：目的 :构建新型高效真核表达载体 ,实现人源化鼠抗人纤维蛋白单链抗体 低分子量尿激酶 (IIn UK)在CHO细胞中的高表达。方法 :利用常规分子生物学方法 ,通过对neo基因的表达进行不同程度的弱化 ,构建了一系列新型真核表达载体 ,以IIn UK融合基因为目的基因 ,比较这些真核表达载体实现外源基因高表达的能力 ,然后挑选高表达的克隆 ,通过MTX加压扩增 ,以提高IIn UK融合基因的拷贝数及表达水平。结果与讨论 :构建了 5种新型真核表达载体 ,并筛选出优化的真核表达载体 ,通过MTX加压扩增 ,实现了IIn UK融合基因在CHO细胞中的高表达 ,表达量达到 10 μg/ (10 6细胞·2 4h )。Objective: To construct new mammalian expression vectors, and to realize the high level expression of humanized scFv-low molecular urokinase(IIn-UK) fusion gene in CHO cell. Methods: By using routine methods of molecular biology, the expression level of neo gene was weakened to different degrees, and the vectors with different neo genes as selection marker were evaluated by comparing the expression level of recombinant IIn-UK fusion gene. To increase the expression level of IIn-UK fusion gene, some clones overexpressing IIn-UK fusion protein were subjected to 2 round amplification with MTX. Results and Conclusion: Five new mammalian expression vectors were constructed, and one most effective mammalian expression vector was confirmed. The CHO cell line that could overexpress IIn-UK fusion protein was obtained, and the expression level reached 10 μg/(10 6cell·24 h).

关 键 词：真核表达载体 neo基因 CHO/dhfr^-细胞 重组蛋白 高表达 

分 类 号：R346[医药卫生—基础医学]