Increased Expression Level of Human Blood Clotting Factor VIII Using NS0 Cell Line as a Host Cells.

IF 1.6 4区 生物学 Q4 BIOTECHNOLOGY & APPLIED MICROBIOLOGY
Mahboobeh Zarei, Elaheh Ferdosi-Shahandashti, Mohsen Badalzadeh, Gholam Ali Kardar
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引用次数: 0

Abstract

Background: Coagulation factor VIII (FVIII) is applied for spontaneous hemorrhaging inhibition and excessive bleeding after trauma in patients with hemophilia A. High-quality human recombinant factor VIII (rFVIII) has been produced relatively in large quantities in cultured mammalian cells. NS0 is one of the most common mammalian cell lines for therapeutic protein production. Production of rFVIII has increased due to low FVIII expression levels and rising demand for hemophilia A prophylactic treatment. Several methods have been developed to prevent cell cycle progression in mammalian cells for increased recombinant protein yields.

Objective: The aim of the study was to investigate the level of recombinant BDD-FVIII expression in NS0 mouse myeloma cells. Additionally, the study aimed to determine the effects of chemical drugs, Mitomycin C, Lovastatin, and Metformin on the secretion of FVIII through cell cycle arrest.

Materials and methods: We cultured NS0 cells and transfected them with the 2 μg pcDNA3-hBDDFVIII plasmid by Lipofectamine 3000. The cells were treated with 10 μg.mL-1 Mitomycin C, 20 μM Lovastatin, and 20 mM Metformin separately. After 24 and 48 hours, the samples were collected and, protein expression was analyzed using RT-PCR, Dot blot, and ELISA.

Results: A higher protein expression level was observed in treated cells 24h and 48h after treatment with all three drugs. According to real-time PCR, Metformin treatment resulted in the highest expression level within 24 h (P=0.0026), followed by Mitomycin C treatment within 48 h (P=0.0030).

Conclusion: The NS0 cell line can be regarded as a suitable host for FVIII production. FVIII protein expression level was increased by using Lovastatin, Metformin, and Mitomycin C drugs. Further investigations are suggested, and the potential application of these drugs to increase recombinant protein yield can be used to produce therapeutic proteins in the industry.

以 NS0 细胞系为宿主细胞提高人凝血因子 VIII 的表达水平
背景:凝血因子 VIII(FVIII)可用于抑制血友病 A 患者的自发性出血和外伤后出血过多。NS0 是生产治疗蛋白最常见的哺乳动物细胞系之一。由于 FVIII 表达水平低以及对 A 型血友病预防性治疗的需求不断增加,rFVIII 的产量也在增加。目前已开发出多种方法来防止哺乳动物细胞的细胞周期进展,以提高重组蛋白的产量:本研究旨在调查重组 BDD-FVIII 在 NS0 小鼠骨髓瘤细胞中的表达水平。此外,该研究还旨在确定化学药物、丝裂霉素 C、洛伐他汀和二甲双胍通过细胞周期停滞对 FVIII 分泌的影响:培养 NS0 细胞,用 Lipofectamine 3000 转染 2 μg pcDNA3-hBDDFVIII 质粒。分别用 10 μg.mL-1 丝裂霉素 C、20 μM 洛伐他汀和 20 mM 甲福明处理细胞。24 小时和 48 小时后,收集样本并使用 RT-PCR、Dot 印迹和 ELISA 分析蛋白质表达:结果:在使用三种药物处理细胞 24 小时和 48 小时后,均观察到较高的蛋白质表达水平。根据实时 PCR,二甲双胍处理后 24 小时内的表达水平最高(P=0.0026),其次是丝裂霉素 C 处理后 48 小时内的表达水平(P=0.0030):结论:NS0 细胞系可被视为生产 FVIII 的合适宿主。使用洛伐他汀、二甲双胍和丝裂霉素 C 可提高 FVIII 蛋白表达水平。建议进一步研究,这些药物在提高重组蛋白产量方面的潜在应用可用于工业生产治疗蛋白。
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来源期刊
Iranian Journal of Biotechnology
Iranian Journal of Biotechnology BIOTECHNOLOGY & APPLIED MICROBIOLOGY-
CiteScore
2.60
自引率
7.70%
发文量
20
期刊介绍: Iranian Journal of Biotechnology (IJB) is published quarterly by the National Institute of Genetic Engineering and Biotechnology. IJB publishes original scientific research papers in the broad area of Biotechnology such as, Agriculture, Animal and Marine Sciences, Basic Sciences, Bioinformatics, Biosafety and Bioethics, Environment, Industry and Mining and Medical Sciences.
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