Baculovirus expression system
A technology of recombinant baculovirus and expression cassette, applied in the direction of virus, viral peptide, virus/phage, etc., can solve the problem of reducing the recovery rate of AAV particles
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Embodiment 1
[0113] Sf9 cells (Expression Systems, Davis, CA) were cultured in Corning storage flasks at 28°C in ESF921 or ESF AF medium (Expression Systems) supplemented with 100 units / mL penicillin and 100 μg / mL chain Mycin (HyClone, Logan, UT). Once the cell density reaches approximately 8x10 6 cells / mL for maintenance, the cells were split 1:4.
Embodiment 2
[0115] Deletion of the v-cath gene from the baculovirus backbone
[0116] The protocol of the λred system (Thomason, Sawitzke et al., 2014) was used to perform the deletion. Briefly, a 1.1 kB fragment containing the chloramphenicol acetyltransferase (CAT) expression cassette flanked by two FRTs ( figure 2 , bases 51-1066) were amplified by PCR using primers 2846 (SEQ ID NO: 1) and 6281 (SEQ ID NO: 2) and plasmid pKD3 (The ODIN, Oakland, CA) as templates. The 2.2Kb fragment containing the GFP expression cassette ( figure 2, bases 1067-3223) were amplified by PCR using primers 5701 (SEQ ID NO: 3) and 6282 (SEQ ID NO: 4) and V376 as templates. After gel purification, the two PCR fragments were ligated with primers 6298 (SEQ ID NO:5) and 6299 (SEQ ID NO:6) to form a 3275-bp PCR fragment. The PCR fragment was digested with the restriction enzyme DpnI (New England Biolabs, Ipswich, MA) to remove contaminating plasmid template. The plasmid pKD46 containing red recombinase (The ...
Embodiment 3
[0119] Recombinant baculovirus production
[0120] Recombinant baculoviruses containing the gene of interest were generated using a recombinant baculovirus shuttle vector (bacmid) that can be recombined with a donor plasmid according to the manufacturer's protocol (Invitrogen, Carlsbad, CA). Briefly, the donor plasmid was diluted to 2 ng / μL in sterile TE buffer (10 mM Tris-HCL, 1 mM EDTA, pH 8.0), and 2 μL of the diluted plasmid DNA was used to transform 20 μL of DH10Bac-wild-type (wt )- or DH10Bac-Δv-cath-competent cells. After 2 days of incubation on LB-agar plates at 37°C, white colonies were picked and minipreps of bacmid DNA were prepared according to the manufacturer's protocol (Invitrogen).
[0121] Miniprep bacmid DNA was then used to transfect Sf9 cells to produce recombinant baculovirus according to the manufacturer's protocol (Invitrogen) with modifications. Briefly, 5 μg of miniprep bacmid DNA and 5 μl of GenJet reagent (SignaGeneLabs, Rockville, MD) were each ...
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