1、Infect MA104 cells at a high MOI(3–10PFU/cell)with trypsin-activated virus.
2、Wash the cell sheet several times with serum-free medium following adsorption.
3、Maintain the cells in trypsin-free medium containing 1μg/mL aprotinin or 0.5μg/mL leupeptin.
4、Harvest the cells once the CPE has reached 70–80% (24 h post infection).
Purification of Triple-Layered Virions
1、Combine the lysate with an equal volume of trichlorotrifluoroethane, and homogenize the mixture with a Sorvall Omni-mixer for three 1min cycles on ice.
2、Centrifuge the homogenates at low speed to separate the organic and aqueous phases (4100g for 10 min in a Sorvall GSA rotor at 4°C).
3、Pellet the virus particles from the aqueous phase by centrifugation at 90,000g in a Beckman (Palo Alto, CA) SW28 rotor or at 45,000g in a Beckman Type 19 rotor for 2h at 4°C.
4、Resuspend the pellet in TBS, and add CsCl to the virus suspension to achieve a density of 1.370g/cm3, as determined by refractometry (see Note 7).
5、Centrifuge the mixture at 110,000g in a Beckman SW50.1 rotor for 20h at 4°C.
6、Inspect the gradients in a darkened room with an inverted light source, to reveal two bands.
7、Recover the virus from the gradient by puncturing the bottom of the centrifuge tube with a 21-gage needle, and collect drops containing TLPs and DLPs, respectively,into separate tubes.
8、Remove the CsCl by dialyzing the virus extensively against TBS at 4°C.
9、Store the dialyzed samples at 4°C in the presence of 0.01% NaN3.
Tris-buffered saline(TBS): 8g NaCl, 0.38g KCl, 0.1g Na2HPO4, 1g dextrose,3g Tris-base, 0.1g MgCl2, 0.1g CaCl2. Dissolve in distilled H2O, adjust to pH 7.4 with HCl, and make up to 1L.
