A scalable suspension insect cell transfection method for production of baculoviruses with low amplification passages

Autor(es)

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]

Afiliação do(s) autor(es)

^[1] Univ Fed ABC, Ctr Ciencias Nat & Humanas, Campus Sao Bernardo Campo, Rua Arcturus, 03 Jardim, BR-09606070 Sao Paulo - Brazil

• Tipo de documento: Artigo Científico
• Fonte: METHODSX; v. 7, 2020.
• Citações Web of Science: 0

Resumo

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)

• Processo FAPESP: 09/11347-6 - Arboviroses emergentes na Amazônia Ocidental: diagnóstico sorológico e molecular
• Beneficiário: Marcia Aparecida Speranca
• Modalidade de apoio: Auxílio à Pesquisa - Regular
• Processo FAPESP: 13/26096-4 - Expressão heteróloga da enzima quitinase de Leishmania (L.) infantum chagasi, Leishmania (V.) braziliensis e Leishmania amazonensis: diagnóstico sorológico e estudo molecular comparativo utilizando sistemas de expressão em células de insetos e bactéria
• Beneficiário: Aline Diniz Cabral
• Modalidade de apoio: Bolsas no Brasil - Pós-Doutorado
• Processo FAPESP: 16/14514-4 - Caracterização da quitinase das espécies de Leishmania endêmicas da América do Sul: utilização no diagnóstico em humanos, cães e flebotomíneos.
• Beneficiário: Marcia Aparecida Speranca
• Modalidade de apoio: Auxílio à Pesquisa - Regular
• Processo FAPESP: 12/20221-9 - Diagnóstico molecular e sorológico das espécies de Leishmania em amostras clínicas de pacientes e cães com leishmanose visceral do centro-oeste paulista
• Beneficiário: Marcia Aparecida Speranca
• Modalidade de apoio: Auxílio à Pesquisa - Regular