Experiments on the preparation of baculovirus virus reservoirs

Summary

Arboviruses are a class of double-stranded circular DNA viruses with capsid-encapsulated genomes of 80-180 kb in size that infect and spread in nature using arthropods as specialized hosts.

Operation method

basic program

Materials and Instruments

baculovirus
Fetal Bovine Serum Recombinant Virus
Incubator Flasks Rotors

Move

Prepared from monolayer culture:1a. Cells were inoculated into two 150 ^cm2 culture flasks at a density of 1.8 x ¹⁰⁷ Sf9 cells per flask and cultured in complete culture medium containing 10% fetal bovine serum. Cells were allowed to attach to the wall at 27°C for 3 h, and then the complete culture medium was removed. Add 5 ml of serum-free complete culture medium containing wild-type or recombinant virus at a viral load of 0.1 of the infected plural. The cells were incubated at 27°C for 1 h.2a. Remove the viral inoculum and replace it with 20 ml of complete culture medium with 10% fetal bovine serum and incubate at 27°C for 1 h. Examine the cells daily under a light microscope for the presence of inclusion bodies or cytopathic effects.3a. The virus is harvested when the majority of cells contain inclusion bodies or show cytopathic effects (usually 4 to 5 days after infection). Infected cultures are transferred into sterilized tubes and centrifuged at 1,000 g for 10 min at 4°C. Supernatants are transferred to a new sterilized tube, and small portions of 1 ml of the supernatant are pipetted into several screw-top freezing tubes for long-term storage in liquid nitrogen tanks, and the remaining viral stocks are stored at 4°C in the dark.Prepared from suspension culture grids:1b. Cultivate Sf9 cells in 100 ml rotary flasks with 50 ml of complete culture medium containing 10% fetal bovine serum, shaking continuously until they grow to approximately 2 × ¹⁰⁶ cells/ml. Centrifuge the cells at 1,000 g for 10 min at room temperature to recover the cells and discard the supernatant. The cells were recovered by centrifugation at 1,000 g for 10 min at room temperature, and the supernatant was discarded. The cell clusters were resuspended by adding 10-20 ml of serum-free complete culture medium containing wild-type or recombinant virus at a viral load of 0.1 of the number of infections. Incubate for 1 h at room temperature.2b. Increase the volume to 100 ml with complete culture medium containing 10% fetal bovine serum Place the cells in a 100 ml rotary culture flask and incubate at 27℃ for 3~4 days with constant shaking.3b. When the majority of cells contain inclusion bodies or show cytopathic effects, harvest the infected cells and treat them as described in step 3a.

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