Wu Jieheng, Han Donghui, Wei Ming, Zheng Guoxu, Jiao Dian, Xi Wenjin, Yang Angang, Qin Weijun, Wen Weihong
Xi Bao Yu Fen Zi Mian Yi Xue Za Zhi. 2016 Jan;32(1):1-4.
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.
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 chain 3 ∶ 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.
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 cells. Flow cytometry showed that the expressed antibody could specifically bind to PSMA positive cells.
CHO-S cel s were successful y domesticated from adherent CHO cells, and could be used for antibody expression. This study provided a useful tool for further antibody expression, purification and function study.
将贴壁生长的中国仓鼠卵巢（CHO）细胞驯化为在无血清培养基中培养的悬浮CHO细胞（CHO-S），并评估CHO-S细胞在抗体表达中的应用。
通过悬浮培养并逐渐降低血清浓度，将贴壁CHO细胞驯化为CHO-S细胞，最终使其在无血清培养基中培养。根据从噬菌体展示文库获得的抗前列腺特异性膜抗原（PSMA）单链抗体片段（ScFv）基因序列，设计、合成重链和轻链基因，并克隆到pcDNA3.1载体中，产物分别命名为pcDNA3.1-HC和pcDNA3.1-LC。使用FreeStyle MAX转染试剂将质粒以轻链和重链3∶1的比例瞬时转染到CHO-S细胞中，转染后第7天收集上清液。采用SDS-PAGE和Western印迹法检测抗体表达，应用流式细胞术评估其与PSMA阳性细胞的结合活性。
贴壁CHO细胞成功驯化为CHO-S细胞。成功构建了抗PSMA抗体重链和轻链表达质粒，抗PSMA抗体可在CHO-S细胞中分泌表达。流式细胞术显示，表达的抗体可特异性结合PSMA阳性细胞。
成功从贴壁CHO细胞驯化为CHO-S细胞，且可用于抗体表达。本研究为进一步的抗体表达、纯化及功能研究提供了有用工具。
