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

发布时间：2018-11-23 19:01

【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。
[Abstract]:Objective to acclimate the adherent Chinese hamster ovarian (CHO) cells into suspension CHO cells (CHO-S) cultured in serum-free medium and verify the feasibility of using the suspension cells for antibody expression. Methods after adjusting the adherent CHO-S cells to suspension culture, they were acclimated into CHO-S cells suspended on serum-free medium by gradually decreasing the serum content. According to the gene sequence of anti-prostate specific membrane antigen (PSMA) single-chain antibody screened from phage antibody library, the heavy chain and light chain genes were designed and synthesized and cloned into eukaryotic expression vector pc DNA3.1, respectively. The resulting vectors were named pc DNA3.1-HC and pc DNA3.1-LC. The constructed two vectors were transiently transfected into CHO-S cells by Free Style MAX transfection reagent at a light and heavy chain ratio of 3:1. The supernatants were collected after 7 days of transfection, and the antibody expression was detected by SDS-PAGE and Western blot methods. The binding activity of PSMA positive cells was detected by flow cytometry. Results the adherent CHO cells were successfully acclimated into suspension CHO-S cells, and the eukaryotic expression vectors expressing the heavy and light chain genes of human antibody against PSMA were successfully constructed, and the secreted expression of antibodies was realized in CHO-S cells. Flow cytometry confirmed that it could specifically bind to PSMA positive prostate cancer cells. Conclusion suspension CHO-S cells were successfully acclimated, and CHO-S cells after acclimation could be used for eukaryotic expression of antibodies, which provided a tool for the subsequent purification and functional study of antibodies.
【作者单位】：第四军医大学免疫学教研室;第四军医大学西京医院泌尿外科;唐都医院泌尿外科;
【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)
【分类号】：R392-33

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