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Construction Of Cell Lines Highly Expressing Anti-EGFR Monoclonal Antibody

Posted on:2014-01-11Degree:MasterType:Thesis
Country:ChinaCandidate:N LiFull Text:PDF
GTID:2250330401979695Subject:The vet
Abstract/Summary:PDF Full Text Request
The study intended to construct stable cell lines highly expressing anti-epidermal growth factorreceptor (EGFR) monoclonal antibody using Chinese Hamster Ovary (CHO) cells as host.DNA sequences encoding the heavy chain and light chain of anti-EGFR monoclonal antibody werechemically synthesized, and cloned into an expression vector containing two expression units. Afteroptimizing electroporation parameters, the correct expression of anti-EGFR monoclonal antibody inCHO cells was confirmed by transient transfection. Clone screening was conducted in chemicallydefined medium containing G418, the resulting clones were subjected to MTX amplification to furtherincrease their specific productivity, then subcloned by limiting dilution to obtain stable cell lines. Thebiological activity of the recombinant antibody was analyzed by in vitro assay.The optimized electroporation parameters were as follow:20μg plasmid/6×106cells, squarewave,231V,24ms,1pulse. Transient transfection showed that the recombinant expression vectorcorrectly expressed in CHO cells. The proper G418concentration was determined as1mg/ml whenseeded at250cells/well in96-well plates for clone screening.40plates were seeded for clone screening,after culturing in96-well plates for14days,483clones were obtained, all of which were transferredinto24-well plates for a7-day culture, then the titers were determined, and61clones with titers above2mg/ml were transferred into T25flasks for a5-day culture, titers were determined again, and15cloneswith titers above4.7mg/ml were transferred to shake flasks to culture at120rpm. Among the15clonestransferred to shake flasks,4clones designated as anti-EGFR-12#, anti-EGFR-228#, anti-EGFR-355#and anti-EGFR-426#responded to MTX amplification. The4clones were subcloned after MTXamplification,6subclones designated as anti-EGFR-12#-24, anti-EGFR-228#-1and-38,anti-EGFR-355#-6and-40, anti-EGFR-426#-11were selected, which showed good growth andexpression characteristics for antibody production. Anti-EGFR-355#-6had the highest specificproductivity, it reached a peak viable cell density of10.2x106cells/ml and an accumulated titer of704.2mg/L in batch culture analysis. The recombinant antibody expressed from anti-EGFR-355#-6showed significant inhibitory effect on proliferation of epidermoid carcinoma A431cells in vitro. Thestudy provided high-producing stable cell lines for anti-EGFR antibody production.
Keywords/Search Tags:epidermal growth factor receptor, monoclonal antibody, cell line
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