Domestication of suspension CHO cells and its application in the expression of anti-PSMA antibody
WU Jieheng;HAN Donghui;WEI Ming;ZHENG Guoxu;JIAO Dian;XI Wenjin;YANG Angang;QIN Weijun;WEN Weihong;Department of Immunology,School of Basic Medicine,Xijing Hospital;Department of Urology,Xijing Hospital;Department of Urology,Tangdu Hospital,Fourth Military Medical University;Department of Immunology,School of Basic Medicine,Xijing Hospital,Xi'an 710032;
Objective To domesticate adherent Chinese hamster ovary( CHO) cells into suspension CHO cells( CHO-S)cultured in serum-free medium,and evaluate the application of the CHO-S cells in antibody expression. Methods Adherent CHO cells were domesticated into CHO-S cells through suspension culture and gradually decreasing the serum concentration,and eventually the cells were cultured in serum-free medium. Based on the anti-prostate-specific membrane antigen( PSMA) single chain antibody fragment( Sc Fv) gene sequence obtained from a phage display library,the genes of heavy and light chains were designed,synthesized and cloned into the pc DNA3. 1 vector,and the products were named pc DNA3. 1-HC and pc DNA3. 1-LC respectively. The plasmids were transiently transfected at the ratio of light and heavy chain3 ∶ 1 into CHO-S cells using Free Style MAX transfection reagent,and the supernatants were harvested at day 7 after transfection. SDS-PAGE and Western blotting were used to detect the antibody expression,and flow cytometry was applied to evaluate its binding activity to PSMA positive cells. Results The adherent CHO cells were successfully domesticated into CHO-S cells. Expression plasmids for anti-PSMA antibody heavy chain and light chain were successfully constructed,and anti-PSMA antibody could be secretively expressed in CHO-S cel s. Flow cytometry showed that the expressed antibody could specifical y bind to PSMA positive cel s. Conclusion CHO-S cel s were successful y domesticated from adherent CHO cel s,and could be used for antibody expression. This study provided a useful tool for further antibody expression,purification and function study.