[Significantly improvement of antibody expression level in CHO cells through downregulation of the DHFR gene in expression vector].
OBJECTIVE
To increase the expressed level of a human-mouse chimeric antibody against human bladder tumor in dihydrofolate 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 microg/(10(6) cells.24 h) was obtained by subcloning. More than 100 microg/(10(6) cells.24 h) was achieved by zinic ion induction.


CONCLUSIONS
MTX induced-increase of recombinant antibody production in CHO cells can be increased by weakening the expression of DHFR gene.
Bai
Yin
Y
Central Laboratory, Naval General Hospital, Beijing 100037, China.
Wang
Yan
Y
Zhang
Hai-rong
HR
Zhou
Li-jun
LJ
Lu
Ying-qian
YQ
Yu
Li-zhang
LZ
D000818:Animals; D000911:Antibodies, Monoclonal / Q000096:biosynthesis* / Q000235:genetics; D000912:Antibodies, Neoplasm / Q000096:biosynthesis* / Q000235:genetics; D016466:CHO Cells; D003001:Cloning, Molecular; D006224:Cricetinae; D003412:Cricetulus; D015536:Down-Regulation; D005822:Genetic Vectors / Q000235:genetics; D008727:Methotrexate / Q000494:pharmacology; D009154:Mutation; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis*; D013762:Tetrahydrofolate Dehydrogenase / Q000096:biosynthesis* / Q000235:genetics; D014162:Transfection; D001749:Urinary Bladder Neoplasms / Q000276:immunology*
D000911:Antibodies, Monoclonal; D000912:Antibodies, Neoplasm; D011993:Recombinant Fusion Proteins; D013762:Tetrahydrofolate Dehydrogenase; D008727:Methotrexate