悬浮驯化CHO细胞并用于抗前列腺特异性膜抗原的抗体表达  

Domestication of suspension CHO cells and its application in the expression of anti-PSMA antibody

作　　者：吴介恒[1] 韩东晖[2] 魏铭[3] 郑国旭[1] 焦点 席文锦[1] 杨安钢[1] 秦卫军[2] 温伟红[1] 

机构地区：[1]第四军医大学免疫学教研室,陕西西安710032 [2]第四军医大学西京医院泌尿外科,陕西西安710032 [3]唐都医院泌尿外科,陕西西安710038

出　　处：《细胞与分子免疫学杂志》2016年第1期1-4,9,共5页Chinese Journal of Cellular and Molecular Immunology

基　　金：国家重点基础研究发展计划(973计划)(2013CB530500);国家自然科学基金(81172146)

摘　　要：目的将贴壁状态的中国仓鼠卵巢(CHO)细胞驯化成用无血清培养基培养的悬浮CHO细胞(CHO-S),并验证用该悬浮细胞进行抗体表达的可行性。方法将贴壁状态的CHO-S细胞调整为悬浮培养后,再用逐步降低血清含量的方法将其驯化成用无血清培养基悬浮培养的CHO-S细胞。根据从噬菌体抗体库筛选获得的抗前列腺特异性膜抗原(PSMA)单链抗体的基因序列,人工设计并合成其重链和轻链基因,分别克隆入真核表达载体pc DNA3.1中,所得载体命名为pc DNA3.1-HC和pc DNA3.1-LC。将构建好的2个载体按照轻、重链比例为3∶1的量用Free Style MAX转染试剂瞬时转染CHO-S细胞,转染7 d后收集培养上清,用SDS-PAGE和Western blot法检测抗体表达情况,用流式细胞术检测其与PSMA阳性细胞的结合活性。结果成功将贴壁CHO细胞驯化成悬浮CHO-S细胞,成功构建了表达抗PSMA人源性抗体重链和轻链基因的真核表达载体,并在CHO-S细胞中实现了抗体的分泌表达,流式细胞术检测证实其可特异性结合PSMA阳性前列腺癌细胞。结论成功驯化获得悬浮CHO-S细胞,驯化后的CHO-S细胞可用于抗体的真核表达,为后续抗体的大量表达纯化及功能研究提供了工具。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.

关 键 词：细胞驯化 CHO细胞 抗体表达 真核表达 转染 

分 类 号：R392-33[医药卫生—免疫学] Q786[医药卫生—基础医学]