Purification experiments of wild-type baculovirus
Purification experiments of wild-type baculovirus
The baculoviruses are a class of DNA viruses that exclusively infect arthropods in nature. The virus particles are rod-shaped, the genome is a double-stranded circular DNA molecule, and the DNA is compressed and packaged in the form of superhelix in the rod-like capsid, with the size ranging from 90 to 180 Kb. Currently, baculoviruses are widely used in pest control as highly efficient and safe pollution-free biological insecticides.
Operation method
basic program
Materials and Instruments
baculovirus Move 1. Inoculate Sf9 cells at a density of 1. 8x107 cells per 1. 8x107 cells in at least 10 150 cm2 culture flasks. incubate at 27°C for about 2 h, allow the cells to adhere firmly to the wall, and infect with wild-type AcMNPV at an infection multiplicity of 0.1. When it was observed that the majority of cells contained inclusion bodies, the viral supernatant (approximately 200 ml) was transferred to four 50 ml conical tubes.2. Centrifuge at 1 000 g at 4°C for 10 min and pour the viral supernatant into 4 new tubes. Repeat centrifugation to remove all residual cells.3. Place the viral supernatant in an SW-27 ultracentrifuge tube, equilibrate and centrifuge at 4°C, 100 000 g for 30 min to collect the virus. Pour out the supernatant, invert the tube onto a Kimwipe paper towel and pour the liquid as dry as possible. For more product details, please visit Aladdin Scientific website.
TE Proteinase K N-dodecyl sarcosine Phenol Chloroform Isopentanol Ethanol Sodium acetate
Culture flasks Conical flasks Rotors Centrifuges Polystyrene tubes
4. Carefully examine the virus precipitate block and if the virus precipitate is pure (it should have an opaque, whitish appearance), go to step 10 and continue. In most cases, the virus precipitate block will be yellow due to the presence of cellular debris. If this is the case, isolate the virus from the cellular debris using one of the following two methods.Isolation and purification of the virus clumps using a sucrose gradient:5a. Add 1 ml of 0.1xTE buffer and resuspend the virus precipitate by repeatedly blowing up and down. If the precipitate is difficult to resuspend, incubate at 4°C overnight.
6a. Prepare two linear gradients from 25% to 56% sucrose in ultra-pure sucrose dissolved in 0.1 xTE buffer that has been filtered for sterilization in ultracentrifuge tubes.
7a. Carefully spread 0.5 ml of virus suspension to the top of each sucrose gradient and centrifuge at 100 000 g at 4°C for 90 min.
8a. Transfer the viral bands to SW-41 ultracentrifuge tubes using a Pasteur pipette with 0.1 x TE buffer added to the top of the tube.
9a. Centrifuge at 100 000 g for 30 min at 4°C, collect the virus, discard the supernatant, and invert the tube onto a Kimwipe paper towel, pouring the liquid as dry as possible.Microcentrifuge to purify the viral precipitate block:5b. Resuspend the viral precipitate by adding 3 ml of extraction buffer and repeatedly blowing up and down. If resuspension is difficult, incubate at 4°C overnight.6b. Transfer 1.5 ml of virus suspension to two 1.5 ml microcentrifuge tubes. Microcentrifuge at maximum speed for 5 min and transfer the supernatant to a 15 ml polystyrene centrifuge tube.
7b. Resuspend each clump with 1 ml Extraction Buffer and wash.8b. Microcentrifuge at maximum speed for 5 min and combine both supernatants with the supernatant from the 15 ml polypropylene tube.
9b. Make up to 9 ml with Extraction Buffer and transfer to two 15 ml polypropylene centrifuge tubes in 4.5 ml portions. Go to Step 11 to continue.10. Resuspend the precipitate with 9 ml of Extraction Buffer and transfer to two 15 ml polypropylene centrifuge tubes in 4.5 ml aliquots. 11.
11. Add 200 ug of 10 mg/ml Proteinase K to each tube and hold at 50°C for 1 to 2 h.12. Add 0.5 ml of 10% sodium N-dodecyl sarcosinate to each tube and incubate at 50°C for 2 h or overnight.13. Extract the DNA twice with an equal volume of 25:24 : 1 phenol/chloroform/isoamyl alcohol.14. Transfer the aqueous phase containing DNA to another 15 ml polypropylene tube using a wide mouth (5-10 ml) pipette. Add 10 ml of 100% ethanol, invert the tube several times, mix gently, and leave at -80°C for 10 min.
15. Centrifuge at 4°C for 20 min at 1,500 g in a benchtop centrifuge, discard supernatant, wash DNA precipitate with 70% ethanol, and air-dry for 30-60 min. add 800 ul of 1xTE buffer to resuspend the precipitate.
16. 400 ul of each was transferred to two microcentrifuge tubes, and 40 ul of 3.0 mol/l sodium acetate and 2 times the volume of 100% ethanol were added to each tube to reprecipitate the DNA for 10 min.
17. Microcentrifuge for 10 min. discard the supernatant. Wash the DNA precipitate with 70% ethanol and lyophilize, resuspend the DNA in 0.3-1 ml 1xTE buffer. measure the absorbance at 260 nm and calculate the yield. store the DNA at 4°C.