| Full text | |
| Author(s): |
Cabral, Aline Diniz
[1]
;
Garcia, Felipe Baena
[1]
;
da Costa, Renata Torres
[1]
;
Pereira Vasconcelos, Ligia Marinho
[1]
;
Uehara, Mabel
[1]
;
Santos, Edmar Silva
[1]
;
Speranca, Marcia Aparecida
[1]
Total Authors: 7
|
| Affiliation: | [1] Univ Fed ABC, Ctr Ciencias Nat & Humanas, Campus Sao Bernardo Campo, Rua Arcturus, 03 Jardim, BR-09606070 Sao Paulo - Brazil
Total Affiliations: 1
|
| Document type: | Journal article |
| Source: | METHODSX; v. 7, 2020. |
| Web of Science Citations: | 0 |
| Abstract | |
Baculovirus expression vector systems (BEVS) have been widely used for production of recombinant proteins in insect cells. However, baculoviruses superinfection and repeated passages originate defective interfering particle (DIP) mutants, which is a limitation to a continuous large-scale production. Accordingly, a classical chemical transfection method performed on monolayer of Spodoptera frugiperda insect cells (Sf9) was modified to produce recombinant baculoviruses with high efficiency. Modifications consist to transfect exponentially growing cells in suspension after concentration by tenfold through centrifugation. Ten different constructions of recombinant baculoviruses with insert varying in size from 180 bp to 2,395 bp, were obtained through employment of the Bac-to-Bac expression system (ThermoFisher/Invitrogen). The transfection efficiency of the modified protocol varied from 45 to 57%, independent of the insert size, while classical method present transfection efficiency of 2 to 20%. After transfection of 6 x 10(6) cells, the recombinant baculoviruses titer obtained with modified method was about 2 x 10(7) pfu/mL in a total volume of 12 mL, which is scalable to 24 liters of 1 x 10(8) pfu/ mL, after only two amplification rounds, contributing to improve large scale heterologous protein production in insect cells, with low amplification passages. (C) 2020 The Author(s). Published by Elsevier B.V. (AU) | |
| FAPESP's process: | 09/11347-6 - Emergent arboviruses in ocidental Amazon: molecular and sorologic diagnosis |
| Grantee: | Marcia Aparecida Speranca |
| Support Opportunities: | Regular Research Grants |
| FAPESP's process: | 13/26096-4 - Heterologous expression of the chitinase enzyme of Leishmania (L.) infantum chagasi, Leishmania (V.) braziliensis and Leishmania (V.) amazonensis: serological diagnosis and comparative molecular study using expression systems of insects cell and bacteria |
| Grantee: | Aline Diniz Cabral |
| Support Opportunities: | Scholarships in Brazil - Post-Doctoral |
| FAPESP's process: | 16/14514-4 - Characterization of the chitinase from South American endemic Leishmania species: use in diagnosis in humans, dogs and sandflies. |
| Grantee: | Marcia Aparecida Speranca |
| Support Opportunities: | Regular Research Grants |
| FAPESP's process: | 12/20221-9 - Molecular and serological diagnostic of Leishmania species in clinical samples of patients and dogs from the Midwest of São Paulo, Brazil, presenting visceral leishmaniasis |
| Grantee: | Marcia Aparecida Speranca |
| Support Opportunities: | Regular Research Grants |