Early Detection of Productive Baculovirus DNA Transfection

Expression of functional Anopheles merusα‐amylase in the baculovirus/Spodoptera frugiperda system

“…Sf 9 insect cells were transfected with the Bd‐AmerAmy, Bd‐ZsAmerAmy and Bd‐ZsAmy bacmid constructs for primary baculovirus particle production. After transfection, baculovirus production was confirmed by two different methods, based on the work we previously reported (Folgueras‐Flatschart et al ., 2000), namely the observation of the cytopathic effect that occurs upon subculturing of transfected cells and amplification of specific DNA fragments, by PCR, using culture supernatants potentially containing baculovirus particles, as a template and specific primers for AmerAmy‐F, ZsAmerAmy‐F and AmerAmy‐R. Baculovirus master stocks were amplified for Sf 9 infection and protein production.…”

“…We previously used the baculovirus/insect cell expression system to produce active mouse Fos and Jun proteins (Corvello et al ., 1995), bovine Herpesvirus glycoproteins (Folgueras‐Flatschart et al ., 2000), the alpha7 subunit of acetylcholine nicotinic receptor (Aztiria et al ., 2000) and human prolactin (Pereira et al ., 2001). Here we describe the results showing that, for the first time, it was possible to express the A. merus α‐amylase in the active form.…”

“…The culture medium from each transfection was collected, clarified (500 g for 5 min) and stored at 4 °C as a master virus stock. Transfection efficiency, recombinant baculovirus (Bv‐AmerAmy, Bv‐ZsAmerAmy and Bv‐ZsAmy) and nonrecombinant baculovirus (Bv) production were monitored by visualization of the cytopathic effect displayed by transfected cells within 48 h after subculturing, under a phase contrast microscope and assaying the presence of baculovirus DNA through PCR analysis (Folgueras‐Flatschart et al ., 2000). To this end, baculovirus present in 50 µl of infected culture supernatant was sedimented at 12 000 g for 10 min in a microcentrifuge tube, and a volume (25 µl) of proteinase K buffer (10 m m Tris‐HCl, pH 7.8; 5 m m EDTA; 0.5% SDS) containing 50 µg/ml of proteinase K (Sambrook et al ., 1989) was added to the pellet to digest viral proteins during 1 h at 56 °C.…”

NUCEL (Cell and Molecular Therapy Center): A Multidisciplinary Center for Translational Research in Brazil

“…Our previous experience with recombinant proteins production using the BEV system [38][39][40][41][42][43] prompted us to subclone the cDNAs to several of the proteins described above in the pFastBac baculovirus vector (Invitrogen) for expression in the S f 9 Spodoptera frugiperda insect cell line. Recombinant bacmids have been obtained for human FSH a and b subunits, FVIII light and heavy chains, and BMPs 2 and 7.…”

Optimized Insect Cell Culture for the Production of Recombinant Heterologous Proteins and Baculovirus Particles

“…One of the most widely used tools for the production of large amounts of recombinant proteins is the Autographa californica multicapsid nuclear polyhedrosis virus (AcMNPV), employed as an expression vector for foreign genes. The baculovirus expression system has been developed and utilized to produce foreign proteins in insect cells (3,5,8,14,17,19). Using recombinant baculovirus vectors based on AcMNPV and Spodoptera frugiperda (Sf9) insect cells, it has been possible to produce secreted proteins, transmembrane proteins, and nuclear proteins.…”