摘要
目的:提高抗人膀胱癌人-鼠嵌合抗体在二氢叶酸还原酶缺陷的CHO细胞株(CHO/DHFR-)中表达的产量。方法:对表达载体中DHFR基因表达调控序列进行一系列的删除突变和点突变后,构建不同的嵌合抗体表达载体。用这些载体转染CHO细胞并以递增量的氨甲喋呤(MTX)筛选,用ELISA法测定每个加压水平的各组细胞中抗体的表达水平。结果:通过对表达载体中DHFR基因表达调控序列的突变,使DHFR的基础表达水平产生了不同程度的弱化。转染CHO细胞后,可明显提高MTX加压增加抗体表达的效果,抗体表达水平的增加基本与DHFR基因的弱化程度呈正相关。从弱化程度最高的pWS2-BDI组中,我们筛选得到表达量达55μg/(106细胞·24 h)的高表达细胞株,经锌离子进一步诱导后,表达量可达100μg/(106细胞·24 h)以上。结论:弱化表达载体中DHFR的表达,可提高MTX对工程抗体表达的增加效果。
AIM: To increase the expressed level of a human-mouse chimeric antibody against human bladder tumor in dihydrofo-late reductase (DHFR) defective CHO cells(CHO/DHFR) via weakening the transcription of DHFR gene in the vector. METHODS: A series of chimeric antibody expression vectors with different deletions and mutations in the modulator sequence of DHFR gene were constructed to downregulate the DHFR gene expression. The vectors were used to transfect CHO/DHFR cells and the transfected cells were subjected to gene amplification in medium containing gradually increasing methotrexate (MTX). The expressed chimeric antibody was quantitated by ELISA. RESULTS: The downregulation of vector-produced DHFR gene introduced by mutation of the modulator sequence could significantly improve the gene amplification effect and the increased antibody production correlated to the reduction of DHFR gene expression. From the best group, a clone with antibody production of 55 ug/( 106 cells ?24 h) was obtained by subclon-ing. More than 100 ug/( 106 cells ?24 h) was achieved by zinic ion induction. CONCLUSION: MTX induced-increase of recombinant antibody production in CHO cells can be increased by weakening the expression of DHFR gene.
出处
《细胞与分子免疫学杂志》
CAS
CSCD
北大核心
2003年第1期62-64,67,共4页
Chinese Journal of Cellular and Molecular Immunology
基金
国家自然科学基金资助(No.30070706)
关键词
载体
DHFR基因
真核表达
抗体
CHO细胞
chimeric antibody
dihydrofolate reductase
CHO cells
eukaryotic expression
