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

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dc.contributorLaboratório de Imunologia Viralpt_BR
dc.contributor.authorRezende, Alexandre Gonçalves dept_BR
dc.contributor.authorNúñez, Eutimio Gustavo Fernándezpt_BR
dc.contributor.authorAstray, Renato Mancinipt_BR
dc.contributor.authorPuglia, Ana Lia Pradellapt_BR
dc.contributor.authorPereira, Carlos Augustopt_BR
dc.contributor.authorJorge, Soraia Attie Calilpt_BR
dc.identifier.citationRezende AG, Núñez EGF, Astray RM, Puglia ALP, Pereira CA, Jorge SAC. An optimization study for expression of the rabies virus glycoprotein (RVGP) in mammalian cell lines using the Semliki Forest virus (SFV). J biotechnol. 2019 Oct;304:63-69. doi:10.1016/j.jbiotec.2019.08.012.pt_BR
dc.description.abstractThe 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.pt_BR
dc.description.sponsorshipFundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)pt_BR
dc.description.sponsorshipConselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)pt_BR
dc.description.sponsorshipCoordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)pt_BR
dc.publisherElsevier Science Publisherspt_BR
dc.relation.ispartofJournal of Biotechnologypt_BR
dc.rightsOpen Accesspt_BR
dc.titleAn optimization study for expression of the rabies virus glycoprotein (RVGP) in mammalian cell lines using the Semliki Forest virus (SFV)pt_BR
dc.contributor.externalUniversidade de São Paulo (USP)¦¦Brasilpt_BR
dc.relation.ispartofabbreviatedJ biotechnolpt_BR
dc.identifier.citationabntv. 304, p. 63-69, oct. 2019pt_BR
dc.identifier.citationvancouver2019 Oct;304:63-69pt_BR
dc.contributor.butantanAstray, Renato Mancini|:Pesquisador|:Laboratório de Imunologia Viral|:pt_BR
dc.contributor.butantanPuglia, Ana Lia Pradella|:Aluno|:Laboratório de Imunologia Viral|:pt_BR
dc.contributor.butantanPereira, Carlos Augusto|:Pesquisador|:Laboratório de Imunologia Viral|:pt_BR
dc.contributor.butantanJorge, Soraia Attie Calil|:Pesquisador|:Laboratório de Imunologia Viral|:Autor de correspondênciapt_BR
dc.contributor.butantanRezende, Alexandre Gonçalves de|:Aluno|:Laboratório de Imunologia Viral|:PrimeiroAutorpt_BR
dc.sponsorship.butantanConselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)¦¦152538/2012-7pt_BR
dc.sponsorship.butantanCoordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)¦¦pt_BR
dc.sponsorship.butantanFundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)¦¦2009/08038-1pt_BR
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