Laboratório de Imunologia Viral, Instituto Butantan, São Paulo, SP, Brazil.
Laboratório de Células Animais, Departamento de Engenharia Química, Escola Politécnica, Universidade de São Paulo, São Paulo, SP, Brazil; Escola de Artes, Ciências e Humanidades (EACH), Universidade de São Paulo, São Paulo, SP, Brazil.
J Biotechnol. 2019 Oct 10;304:63-69. doi: 10.1016/j.jbiotec.2019.08.012. Epub 2019 Aug 20.
The Semliki Forest virus (SFV) viral vector has been widely used for transient protein expression. This study aimed to analyze comprehensively the capacity of SFV vector to express rabies lyssavirus glycoprotein (RVGP) in mammalian cells. The assessed parameters were transfection strategy, multiplicity of infection (MOI), harvest time and mammalian cell host. Two transfection approaches, electroporation and lipofection were evaluated to obtain the recombinant SFV, and the electroporation was found to be the most effective. Viral quantification by RT-qPCR was performed to elucidate the relation between the amount of recombinant virus utilized in the infection process and the production levels of the heterologous protein. Four different multiplicities of infection (MOIs = 1; 10; 15; 50) were evaluated using five mammalian cell lines: BHK-21, HuH-7, Vero, L929, and HEK-293T. Protein expression was assessed at two harvest times after infection (24 and 48 h). The recombinant protein generated was characterized by western blot, dot blot, and indirect immunofluorescence (IIF), while its concentration was determined by enzyme-linked immunosorbent assay (ELISA). Similar expression patterns were observed in cell lines BHK-21, HEK-293T, L929, and Vero, with higher RVGP production in the first 24 h. The BHK-21 cells showed yields of up to 4.3 μg per 10 cells when lower MOIs (1 and 10) were used. The HEK-293 T cells also showed similar production (4.3 μg per 10 cells) with MOI of 1, while the L929 and Vero cell lines showed lower expression rates of 2.82 and 1.26 μg per 10 cells, respectively. These cell lines showed lower expression levels at 48 h after infection compared to 24 h. Controversially, in the case of the HuH-7 cell line, RVGP production was higher at 48 h after infection (4.0 μg per 10 cells) and using MOIs of 15 and 50. This work may contribute to optimize the RVGP production using SFV system in mammalian cells. This study can also substantiate for example, the development of approaches that use of SFV for applications for other protein expressions and suggests values for relevant parameters and cell lines of this biotechnique.
辛德毕斯森林病毒 (SFV) 病毒载体已被广泛用于瞬时蛋白表达。本研究旨在全面分析 SFV 载体在哺乳动物细胞中表达狂犬病溶蛋白糖蛋白 (RVGP) 的能力。评估的参数包括转染策略、感染复数 (MOI)、收获时间和哺乳动物细胞宿主。评估了电穿孔和脂质体两种转染方法来获得重组 SFV，结果发现电穿孔最为有效。通过 RT-qPCR 进行病毒定量，以阐明感染过程中使用的重组病毒数量与异源蛋白产生水平之间的关系。使用 BHK-21、HuH-7、Vero、L929 和 HEK-293T 五种哺乳动物细胞系评估了四种不同的感染复数 (MOI=1；10；15；50)。感染后 24 和 48 小时评估蛋白质表达。通过 Western blot、斑点印迹和间接免疫荧光 (IIF) 检测生成的重组蛋白，通过酶联免疫吸附测定 (ELISA) 测定其浓度。在 BHK-21、HEK-293T、L929 和 Vero 细胞系中观察到相似的表达模式，在最初的 24 小时内产生更高的 RVGP。当使用较低的 MOI (1 和 10) 时，BHK-21 细胞的产量高达每 10 个细胞 4.3μg。HEK-293T 细胞在 MOI 为 1 时也表现出类似的产量（每 10 个细胞 4.3μg），而 L929 和 Vero 细胞系的表达率分别为 2.82 和 1.26μg/10 个细胞。与感染后 24 小时相比，这些细胞系在感染后 48 小时的表达水平较低。相反，在 HuH-7 细胞系中，感染后 48 小时 (4.0μg/10 个细胞) RVGP 的产量更高，MOI 为 15 和 50。这项工作可能有助于优化 SFV 系统在哺乳动物细胞中 RVGP 的生产。本研究还可以为 SFV 用于其他蛋白质表达的应用提供依据，并为该生物技术的相关参数和细胞系提供参考值。
