Navegando por Palavras-chave "Glicoproteína Do Vírus Da Raiva"

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    Padrão de glicosilação da glicoproteína do vírus da raiva produzida em células drosophila melanogaster S2: diferentes sistemas de expressão e condições de cultivo tese
    (Universidade Federal de São Paulo (UNIFESP), 2019-11-13) Silva, Livia Pilatti Mendes Da [UNIFESP]; Augusto, Elisabeth De Fatima Pires [UNIFESP]; Universidade Federal de São Paulo (UNIFESP)
    Rabies virus glycoprotein (RVGP) produced in animal cells had become an attractive alternative for development of a new vaccine against rabies with lower biosecurity risk and lower costs. In recombinant proteins production host cell lineage and cultivation parameters and conditions are important factors for glycoform profile of final protein, which impacts directly in its function. Objectives: Determine and compare the glycosylation pattern of rabies virus glycoprotein (RVGP) expressed in insect Drosophila melanogaster Schneider 2 (S2), in different expression systems and culture conditions. Methods: S2 cells expressing RVGP in cell membrane using inducible (S2MtRVGP-imm) or constitutive (S2AcRVGP-2) promoter, and expressing the soluble RVGP ectodomain, using an inducible promoter (S2BipMt_RVGP-Ecto), stablished in previous studies, were used in the present work. S2BipMt_RVGP-Ecto cells were cultivated in schott flasks and S2MtRVGP-imm cells were cultivated in schott and spinner flasks, in both cases with different induction parameters and cultivation time. S2AcRVGP-2 cells grown in bioreactor, in batch or fed-batch with glutamine supplementation. An immunoaffinity chromatography methodology for membrane rRVGP (rRVGP-S2Mt and rRVGP-S2Ac) were stablished using a commercial monoclonal antibody attached to a HiTrap NHSactivated HP (GE). Soluble ectodomain (rRVGP-S2Ecto) were purified by Immobilized Metal Affinity Chromatography (IMAC-Ni+2). Glycosylation pattern were analyzed by hydrophobic interaction chromatography (HILIC) or by HILIC accoupled with a mass spectrometry (UPLC-FLR-MS/MS). Results: For S2BipMt_RVGP-Ecto cells, the variation in inductor concentration presented biological effect that reflected on cell growth and rRVGP production. Inductor concentration and cell concentration at induction resulted in an increase in rRVGP production speed by S2MtRVGP-imm cells. Cultivation on spinner flasks results in a higher production of rRVGP-S2Mt comparing with schott flasks cultivation. The strategy of low initial glutamine concentration with supplementation during the S2AcRVGP-2 cell cultivation decreased the ammonium formation after the end of exponential phase, with an impact on growth and rRVGP production. Immunoaffinity chromatography methodology purification generated samples with high level of purity and efficiency up to 99 % of trimeric rRVGP-S2Mt. For rRVGP-S2Ac the same methodology worked with less efficiency and low level of purity. IMAC-Ni+2 used for rRVGP-S2Ecto presented low level of purity due to the low amount of expressed glycoprotein. In glycan pattern analyzes, samples of rRVGP-S2Ecto presented in higher proportion paucimannosidic structures: F(6)M2, M3 and F(6)M3. In rRVGP-S2Mt samples, the structures F(6)M2 e M3 were detected in higher proportion. rRVGP-S2Ac samples presented higher proportion of structures M2, F(6)M2 e M3. Conclusions: Increase of inductor concentration and of cell concentration at induction time results in a biological effect, by increasing expression speed of rRVGP. Specific production speed of rRVGP has a direct influence on decrease of N-glycans processing. Furthermore, different cultivation modes resulted in variation in glycan pattern, and a drastic reduction in initial glutamine concentration resulted in an increase on glycoforms with five or more mannose residues.
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    Uso do modo de operação em contínuo para avaliar aspectos fisiológicos de linhagem recombinante de drosophila melanogaster
    (Universidade Federal de São Paulo (UNIFESP), 2019-02-19) Oliveira, Carla Reis [UNIFESP]; Augusto, Elisabeth De Fatima Pires [UNIFESP]; Universidade Federal de São Paulo (UNIFESP)
    Glycoproteins are complex molecules produced preferentially by animal cells, but their quality pattern rely on the knowledge of cell metabolism and requires engineering tools to ensure greater uniformity and productivity. As long as reports on the metabolism of insect cells are scarce, researches on the physiological aspects of Drosophila*melanogaster are of utmost importance to promote their usage. In order to contribute on the knowledge of this metabolism, the present work aimed to identify growth limiting and inhibitory factors of S2AcRVGP2, a D.*melanogaster S2 cell line stably transfected for the synthesis of Rabies virus glycoprotein (GPV). This study was conducted using TC100 based medium in spinner flasks and in benchtop bioreactor stirred at 100 rpm and with bubbleLfree aeration system. Exploratory cultures on batch mode were carried out with different concentrations of LLcystine (CYS2) and magnesium (Mg2+). The influence on the metabolism of different glucose concentrations (GLC) in the feed medium was studied in a continuous mode. It has been shown that lactate and ammonium did not seem to be growth inhibiting. The supplementation with CYS2 11 mg/L enhanced GPV expression by 75% in batch with initial supplementation and 21% in batch with pulse addition at the end of exponential phase. The evaluation of Mg2+ influence on cell growth kinetic parameters showed that when cultivated with half of its basal concentration, cell production increased 37%, but when cultured on [Mg2+] inferior to 121 mg/L, it limited the growth rate and altered cell membrane morphology. Continuous culture with glucose concentration above 3.75 g/L in the feed medium indicated overflow on glycolysis with consequently limitation of cell growth. The metabolism shifted to a more balanced state when fed with glucose concentration under 2.5 g/L. Cell production increased by 40% and GPV expression doubled in the lowest glucose concentration evaluated (1.25 g/L).