Poxvirus purification assay
Poxvirus purification assay
Poxvirus is usually purified by sucrose density gradient centrifugation. The purified virus can be used to prepare poxvirus DNA for use in studies that do not allow for the presence of protein contamination from infected cells, as well as for use as a high-titer virus reservoir. For large-scale purification (at least 1 L of culture), the best approach is to use HeLa cell suspensions as the infected object rather than monolayer cultured cells. Content from Compact Molecular Biology Laboratory Guide (5th Edition)
Operation method
Mass purification
Principle
For many studies, partially purified virus is sufficient (proceed to step 14 of this experiment), and in the case of purified MVA virus, trypsin digestion is not possible, nor can CEF cells or BHK-21 cells be used in place of HeLa cells.
Materials and Instruments
Poxvirus reservoir solution HeLa S3 cells (suspension culture) Move 1. Mix equal volumes of jaundice virus reservoir solution and 0.25 mg/ml trypsin before use, and shake vigorously. incubate at 37℃ for 30 min, and vortex and oscillate every 5-10 min during the incubation process. 2. Count HeLa S3 cells with a hemocytometer (Appendix 3F). 3. Transfer 5 × 108 HeLa S3 cells to a centrifuge tube, centrifuge at 1800 g for 10 min at room temperature, and discard the supernatant. 4. Then resuspend the cells at a concentration of 2 × 107 cells/ml with Spin Shaker Complete Medium-5. 5. Add the digested virus to the cell suspension at 5-8 pfu/cell (MOI) and incubate for 30 min with agitation. 6. Transfer the cells to an aerated rotary shaker containing 1 L of Rotary Shaker Complete Medium-5 and incubate with agitation for 2 to 3 days. 7. 5. ～Centrifuge the cells at 1800 g for 5 min at ~10°C and discard the supernatant. 8. Suspend the cells in 14 ml of 10 mmol/L Tris-C solution pH 9.0. Keep the sample on ice for the following steps. 9. Homogenize the cell suspension with 30-40 strokes of a Doens homogenizer and examine the cells by microscopy for fragmentation. 10. 5. ～Centrifuge at 300 g (900 r/min in H-6000A rotor) for 5 min at ~10°C to remove nuclei and retain supernatant. 11. Suspend the cell precipitate in 3 ml of 10 mmol/L Tris-C solution pH 9.0. Centrifuge at 300 g for 5 min at 5-10°C, retain the supernatant and combine with the supernatant from step 10. 12. Sonicate the supernatant (dissolution product), keeping the dissolution product cold throughout, using a cup sonicator according to the following procedure. 12.1 Dispense the samples in portions of 3 ml each and sonicate them separately. 12.2 Fill the cup with an ice-water mixture (50% ice), place the tube containing the dissolution product in the ice-water mixture and sonicate for 1 min at maximum power. 12.3 Repeat sonication 3 or 4 times, placing the dissolved product on ice for ≥ 30 s after each sonication. 13. Add the solubilized product to a sterile SW27 (or SW28) centrifuge tube containing a 17 ml 36% sucrose cushion. Centrifuge at 32,900 g for 80 min at 4°C. Aspirate the supernatant and discard. 14. 14. Suspend the virus precipitate in 1 ml of 1 mmol/L pH 9.0 Tris-C solution. The virus purified at this point is sufficiently pure for some purposes, such as DNA isolation. 15. Sonicate for 1 min as in step 12. 16. Prepare a sterile 24% to 40% continuous sucrose gradient the day before use by carefully adding 6.8 ml of 40%, 36%, 32%, 28%, and 24% sucrose solution to a sterile SW27 centrifuge tube. Allow to stand overnight in the refrigerator. 17. Add 1 ml of sonicated virus precipitate (from step 15) to the upper layer of the sucrose gradient. centrifuge at 4°C, 26,000 g for 50 min. 18. A milky-white viral band can be seen in the middle of the centrifuge tube. Discard the sucrose solution above the viral band by aspiration and carefully collect the viral band (approximately 10 ml) in a sterile tube with a sterile lance tip for storage. 19. Collect the viral polymers aggregated at the bottom of the sucrose gradient after aspirating the remaining sucrose solution. Resuspend the viral precipitate in 1 ml of 1 mmol/L pH 9.0 Tris -Cl solution by up and down aspiration. 20. Sonicate for 1 min as in step 12. 21. Repeat steps 16 through 18 to collect the viral bands and combine with the viral solution collected in step 18. Mix with two times the volume of 1 mmol/L Tris-Cl solution pH 9.0 and transfer to a sterile SW27 centrifuge tube. The total volume of solution should be 60 ml, enough to fill two SW27 tubes. If a smaller volume is obtained, top up the tubes with 1 mol/L pH 9.0 Tris-C solution. 22. 4 centrifuge at 32,900 g for 60 min and discard the supernatant. 23. 23. Resuspend the virus precipitate in 1 ml of 1 mmol/L pH 9.0 Tris-C solution, sonicate as in Step 12, and dispense 200-250 μl per portion. One portion is retained for step 24 and the rest is frozen at -70°C. 24. Determine the amount of virus required for extraction of poxvirus DNA by quantifying the unfrozen virus sample at 260 nm using a spectrophotometer (see Supporting Scheme 2). 25. Sonicate the viral suspension on ice for 20-30 s (see Step 12) and then make a 10-fold serial dilution up to 10-10. Infect BS-C-1 cells cultured in monolayers in 6-well plates grown to confluence with 3 dilutions, 10-8, 10-9, and 10-10, making a double batch of each dilution. Caveat 1. The titer of the virus reservoir is usually 2 × 109pfu/ml, but the titer of virus reservoirs from different sources may be much lower than this. If visible clumps are present, cool to 0°C and sonicate on ice for 30 s. The sonication may be repeated several times, but the sample should be cooled on ice between sonications. 2. In Step 12. 3， because the process of sonication causes the virus to be removed from the sample, the sample should be cooled on ice. 2. In step 12. 3， ice should be refilled into the cup because the sonication process melts the ice. 3. a unit of optical density is 1.2 × 1010virus particles, i.e., it is (2.5 to 5) × 10 108 pfu.This value may vary slightly from one spectrophotometer to another due to the scattering, non-absorption of light. 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Trypsin Tris - Cl Sucrose
Rotary Shaker Complete Medium-5 2 L aerated rotary shaker with tight pestle and mortar Duanes Homogenizer (with tight pestle) Cup sonicator Beckman Ultracentrifuge Sterile centrifuge tubes (SW27 or SW28 heads) Spectrophotometer