Department of Food Science and Technology, School of Agriculture, Shiraz University, Shiraz, Iran.
Cellular and Molecular Biology Research Center, Shahid Beheshti University of Medical Sciences, Tehran, Iran.
Mol Biol Rep. 2021 May;48(5):4405-4412. doi: 10.1007/s11033-021-06456-0. Epub 2021 Jun 5.
Monoclonal antibodies (mAbs) are widely employed as invaluable therapeutics for a vast number of human disorders. Several approaches have been introduced for the improvement of mAb production in Chinese hamster ovary (CHO) cells due to the increasing demand for these products. In this regard, various chromatin-modifying elements such as insulators have been incorporated in the expression vectors to augment mAb expression. In this study, human gamma-satellite insulator containing vectors were utilized for the expression of an anti-proprotein convertase subtilisin/kexin type 9 (PCSK9) mAb in CHO-K1 cells. To this aim, dual expression vectors encoding the antibody light chain (LC) and heavy chain (HC) with or without the insulator element were constructed, and mAb expression was evaluated in transient and stable expression. Based on the results, mAb expression significantly increased in the stable cell pool, and clonal cells developed using the human gamma-satellite insulator. In contrast, transient antibody expression was not affected by the insulator element. Finally, the enhancement of LC and HC mRNA levels was found in the insulator containing stable cell pools using the quantitative real-time-polymerase chain reaction (qRT-PCR). Our findings showed the positive effect of the human gamma-satellite insulator on the stable expression of an anti-PCSK9 immunoglobulin G1 (IgG1) mAb in CHO-K1 cells using dual expression vectors.
单克隆抗体（mAbs）被广泛用作治疗人类多种疾病的宝贵疗法。由于对这些产品的需求不断增加，人们已经提出了几种方法来提高中国仓鼠卵巢（CHO）细胞中 mAb 的生产。在这方面，已经在表达载体中引入了各种染色质修饰元件，如绝缘子，以提高 mAb 的表达。在这项研究中，使用含有人类γ卫星绝缘子的载体在 CHO-K1 细胞中表达抗蛋白水解酶原转化酶枯草杆菌蛋白酶/kexin 9（PCSK9）mAb。为此，构建了编码抗体轻链（LC）和重链（HC）的双表达载体，带有或不带有绝缘子元件，并在瞬时和稳定表达中评估 mAb 表达。根据结果，mAb 表达在稳定细胞池中显著增加，并且使用人γ卫星绝缘子开发的克隆细胞。相比之下，瞬时抗体表达不受绝缘子元件的影响。最后，使用定量实时聚合酶链反应（qRT-PCR）发现含有绝缘子的稳定细胞池中的 LC 和 HC mRNA 水平增加。我们的研究结果表明，使用双表达载体，人γ卫星绝缘子对 CHO-K1 细胞中抗 PCSK9 免疫球蛋白 G1（IgG1）mAb 的稳定表达具有积极影响。
