Production of Rabies VLPs in Insect Cells by Two Monocistronic Baculoviruses Approach

Bernardino, Thaissa Consoni; Astray, Renato Mancini; Pereira, Carlos Augusto; Boldorini, Vera Lucia Lopes; Antoniazzi, Marta Maria; Jared, Simone G.S.; Núñez, Eutímio Gustavo Fernández; Jorge, Soraia Attie Calil

doi:10.1007/s12033-021-00366-z

Cited by 12 publications

(11 citation statements)

References 52 publications

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“…It is known that co-infection by virus premixture is one of the merits of the baculovirus expression system [ 25 , 28 , 31 , 32 ]. As expected, the additional GST-tag fused to VP15 significantly improved the soluble fraction of VP15 as compared with the relatively low expression level of the VP15-FLAG construct ( Figure 3 B).…”

Section: Resultsmentioning

confidence: 99%

“…To avoid a conflict during protein purification for the FLAG-tagged POIs (GST-VP15-FLAG) from silkworm-BEVS, the enterokinase site (DDDDK) was removed from pET41a-GST-HRV3C using inverse PCR [KOD-Plus-Neo DNA polymerase, (Toyobo, Osaka, Japan); 3Cpt-FW 5′-atgggaccaaacacagaatt-3′ and GGGSG-RV 5′-accggagccaccaccggtaccca-3′]. Subsequently, GST-HRV3C ∆FLAG was amplified using another primer set (GST-FW 5′-atgtcccctatactaggttattgg-3′ and pET-RV 5′-ggttatgctagttattgctc-3′, XholI digested) and inserted into the StuI/XholI double digested pFastbac-1 vector (Thermo Fisher Scientific, K. K.), termed pFastBac-GST-HRV3C, for generating recombinant B. mori nucleopolyhedrovirus (BmNPV) bacmid using BmDH10Bac E. coil [ 32 ]. All of the sequences were confirmed through DNA sequencing.…”

Section: Methodsmentioning

confidence: 99%

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In vivo enzymatic digestion of HRV 3C protease cleavage sites-containing proteins produced in a silkworm-baculovirus expression system

Nakanishi

Kato

et al. 2022

Bioscience Reports

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Baculovirus expression vector system (BEVS) has been recognized as a potent protein expression system in engineering valuable enzymes and vaccines. Various fusion tags facilitate protein purification, leaving the potential risk to influence the target protein’s biological activity negatively. It is of great interest to consider removing the additional tags using site-specific proteases, such as human rhinoviruses (HRV) 3C protease. The current study validated the cleavage activity of 3C protease in Escherichia coli and silkworm-BEVS systems by mixing the cell or fat body lysates of 3C protein and 3C site containing target protein in vitro. Further verification has been performed in the fat body lysate from co-expression of both constructs, showing remarkable cleavage efficiency in vivo silkworm larvae. We also achieved the glutathione-S-transferase (GST) tag-cleaved product of the VP15 protein from the White spot syndrome virus after purification, suggesting that we successfully established a coinfection-based recognition-and-reaction BEVS platform for the tag-free protein engineering.

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Section: Resultsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

In vivo enzymatic digestion of HRV 3C protease cleavage sites-containing proteins produced in a silkworm-baculovirus expression system

Nakanishi

Kato

et al. 2022

Bioscience Reports

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“…Viral batches were obtained by transfection of 10 µg of recombinant RVGP and RVM bacmid complexed with cationic liposome (Cellfectin II ® , Invitrogen, Carlsbad, CA, USA) separately in 5 × 10 6 Sf9 cells, using a 25 cm 2 T-flask in 5 mL of SF900III medium, and culturing them for 96 h. The genetic constructions were defined and implemented as described previously [ 15 ]. Then, the supernatant was clarified and stored at 4 °C and protected from light, giving rise to passage batch 1.…”

Section: Methodsmentioning

confidence: 99%

Performance Comparison of Recombinant Baculovirus and Rabies Virus-like Particles production Using Two Culture Platforms

et al. 2022

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This work aimed to assess, following upstream optimization in Schott flasks, the scalability from this culture platform to a stirred-tank bioreactor in order to yield rabies-recombinant baculovirus, bearing genes of G (BVG) and M (BVM) proteins, and to obtain rabies virus-like particles (VLP) from them, using Sf9 insect cells as a host. Equivalent assays in Schott flasks and a bioreactor were performed to compare both systems and a multivariate statistical approach was also carried out to maximize VLP production as a function of BVG and BVM’s multiplicity of infection (MOI) and harvest time (HT). Viable cell density, cell viability, virus titer, BVG and BVM quantification by dot-blot, and BVG quantification by Enzyme-Linked Immunosorbent Assay (ELISA) were monitored throughout the assays. Furthermore, transmission electron microscopy was used to characterize rabies VLP. The optimal combination for maximum VLP expression was BVG and BVM MOI of 2.3 pfu/cell and 5.1 pfu/cell, respectively, and 108 h of harvest time. The current study confirmed that the utilization of Schott flasks and a benchtop bioreactor under the conditions applied herein are equivalent regarding the cell death kinetics corresponding to the recombinant baculovirus infection process in Sf9 cells. According to the results, the hydrodynamic and chemical differences in both systems seem to greatly affect the virus and VLP integrity after release.

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“…The BVs were purified, first by pelleting through an ultracentrifugation at 200,000 × g for 2 h at 4°C (Sorvall WX80+; Thermo Fisher Scientific, USA). Next, we concentrated the supernatant through a 25% sucrose cushion and then resuspended it in 100 μL PBS ( 41 ). Culture medium of uninfected BmE cells was also purified by this method as a negative control for the experiment.…”

Section: Methodsmentioning

confidence: 99%

The NPC Families Mediate BmNPV Entry

Fan

Bao

et al. 2022

Microbiol Spectr

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Production of Rabies VLPs in Insect Cells by Two Monocistronic Baculoviruses Approach

Cited by 12 publications

References 52 publications

In vivo enzymatic digestion of HRV 3C protease cleavage sites-containing proteins produced in a silkworm-baculovirus expression system

In vivo enzymatic digestion of HRV 3C protease cleavage sites-containing proteins produced in a silkworm-baculovirus expression system

Performance Comparison of Recombinant Baculovirus and Rabies Virus-like Particles production Using Two Culture Platforms

The NPC Families Mediate BmNPV Entry

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