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An optimization study for expression of the rabies virus glycoprotein (RVGP) in mammalian cell lines using the Semliki Forest virus (SFV)

Author:
de Rezende, Alexandre Gonçalves, Fernández Núñez, Eutimio Gustavo, Astray, Renato Mancini, Puglia, Ana Lia Pradella, Pereira, Carlos Augusto, Jorge, Soraia Attie Calil
Source:
Journal of biotechnology 2019 v.304 pp. 63-69
ISSN:
0168-1656
Subject:
Rabies lyssavirus, Semliki Forest virus, T-lymphocytes, Western blotting, cell lines, electroporation, enzyme-linked immunosorbent assay, fluorescent antibody technique, glycoproteins, harvest date, mammals, protein synthesis, quantitative polymerase chain reaction, recombinant proteins, reverse transcriptase polymerase chain reaction, transfection, viruses
Abstract:
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 106 cells when lower MOIs (1 and 10) were used. The HEK-293 T cells also showed similar production (4.3 μg per 106 cells) with MOI of 1, while the L929 and Vero cell lines showed lower expression rates of 2.82 and 1.26 μg per 106 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 106 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.
Agid:
6593376