用反式互补表达载体实现全抗体在CHO-dhfr-细胞中的高效表达  

High Level Expression of Complete Antibody in CHO-dhfr-Cells Using Trans-Complementing Expression Vectors

作　　者：安怀杰[1] 俞炜源[1] 孙志伟[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：《生物技术通讯》2007年第5期735-738,共4页Letters in Biotechnology

基　　金：国家高技术研究发展计划项目(2001AA215351)

摘　　要：目的:通过弱化筛选基因二氢叶酸还原酶基因(dhfr),构建双顺反子全抗体表达载体,实现全抗体在CHO细胞中的高效表达。方法:将dhfr基因分为分别编码1～105和106～187氨基酸残基的2部分,分别通过linker与亮氨酸拉链GCN4融合,分别通过内部起始位点序列与抗体轻重链基因偶联,构建成2个双顺反子表达载体pIRESLZdhfr-H和pIRESLZdhfr-L。用脂质体2000转染CHO-dhfr-细胞,用无次黄嘌呤和胸腺嘧啶脱氧核苷的IMDM筛选培养基筛选阳性克隆。结果:筛选到的阳性克隆表达水平为1.47～3.5μg/(106细胞.d),经氨甲蝶呤(MTX)梯度加压,在MTX浓度为5×10-8mol/L时,表达水平达到11.5μg/(106细胞.d)。结论:构建了基于亮氨酸拉链二聚化基础的dhfr弱化方式的双顺反子表达载体,实现了全抗体在CHO-dhfr-细胞中的高效表达。Objective： To construct complete antibody expression bicistronic vectors via impairing selective marker gene dihydrofolate reductase（dhfr）, and to express complete antibody in CHO-dhfrˉcells at high level. Methods： The dhfr selectable marker can be divided into two fragments（coding 1-105 and 106-187 aa, respectively）. It can also be re-associate to form an active molecule with the aid of a leucine zipper. Using internal ribosome entry site element, two bicistronic expression vectors pIRESLZdhfr-H and pIRESLZdhfr-L were constructed by linking the two antibody chains to the dhfr fragments. The two vectors were then transfected to CHO-dhfr- cells by LipofectAMINE 2000, and selected by selectable IMDM media without hypoxanthine and thymine. Results： The expression level of the initial clones was at 1.47-3.5 μg/ 006 cell·d）. Furthermore, with the presence of methotrexate, expression of each antibody chain in conjunction with a portion of dhfr also leads to concurrent amplification of both antibody chains. At the concentration of MTX 5×10^-8 mol/L, the expression level was improved to 11.5 μg/（10^6 cell-d）. Conclusion： The trans-complementing bicistronic expression vectors via impairment of dhfr re-associated by leucine zipper were constructed. The results show that the high level expression of the complete antibody can be realized at CHO-dhfr- cells.

关 键 词：双顺反子表达载体 二氢叶酸还原酶基因 弱化 全抗体 

分 类 号：Q78[生物学—分子生物学] R392.1[医药卫生—免疫学]