Instituto de Biologia Experimental e Tecnologia/Instituto de Tecnologia Química e Biológica IBET/ITQB -UNL, Apartado 12, 2781-901, Oeiras, Portugal.
Cytotechnology. 2010 Dec;62(6):547-55. doi: 10.1007/s10616-010-9305-x. Epub 2010 Oct 10.
Apoptosis is a major problem in animal cell culture during production of biopharmaceuticals, such as recombinant proteins or viral particles. In the present work baculovirus-insect cell expression system (BEVS/IC) is used as model to produce rotavirus like-particles, composed by three layers of three different viral proteins (VP2, VP6 and VP7). In this model baculovirus infection also induces host cell death. Herein a new strategy to enhance cell life span and to increase recombinant rotavirus protein production of BEVS/IC system was developed. This strategy relies on hemolymph from Lonomia oblique (total extracts or a semi-purified fraction) medium supplementation. The total extract and a purified fraction from hemolymph of Lonomia obliqua were able to protect Sf-9 cell culture against apoptosis triggered by oxidative stress (using the pro-oxidant agents tert butylhydroperoxide and hydrogen peroxide) and by baculovirus infection. Furthermore, hemolymph enhance final recombinant protein production, as it was observed by the increased amounts of VP6 and VP7, which were measured by the semi-quantitative western blot method. In conclusion, hemolymph medium supplementation can be a promising strategy to improve cell viability and productivity of recombinant protein in BEVS/IC system.
细胞凋亡是生物制药生产过程中动物细胞培养的一个主要问题，例如重组蛋白或病毒颗粒。在本工作中，杆状病毒-昆虫细胞表达系统（BEVS/IC）被用作生产轮状病毒样颗粒的模型，这些颗粒由三种不同的病毒蛋白（VP2、VP6 和 VP7）组成的三层组成。在该模型中，杆状病毒感染也会诱导宿主细胞死亡。在此，开发了一种新策略来延长细胞寿命并提高 BEVS/IC 系统中重组轮状病毒蛋白的产量。该策略依赖于美洲钩吻（总提取物或半纯化部分）血淋巴的培养基补充。美洲钩吻的血淋巴的总提取物和半纯化部分能够保护 Sf-9 细胞培养物免受氧化应激（使用促氧化剂叔丁基过氧化物和过氧化氢）和杆状病毒感染引发的细胞凋亡。此外，血淋巴增强了最终的重组蛋白产量，如通过半定量 Western blot 方法测量的 VP6 和 VP7 的增加量所观察到的那样。总之，血淋巴培养基补充可以成为提高 BEVS/IC 系统中细胞活力和重组蛋白产量的有前途的策略。
