2.7. Purification of VEEV-RABV-G virions

Materials

- Cell：BHK-21

-Virus：VEEV-RABV-G

-Dulbecco’s Modified Eagle’s Medium (DMEM; gibco, cat. C11995500BT) supplemented with 10% or 2% fetal bovine serum (FBS, gibco, cat. 10270-106) and 1% AS (Beyotime, cat. C0224-100ml). This is called 10% FBS DMEM or 2% FBS DMEM.

-40% (W/V) PEG8000 (SIGMA, cat.BCCC7539)：40 g PEG8000 was dissolved with PBS (Gbico,cat C10010500BT ) in total volume of 100 mL. Filter sterilize using 0.22 μm filter and store at 4℃. 

 -10% (W/V) Sucrose solution: 1g sucrose was dissolved with PBS (Gbico,cat C10010500BT ) in total volume of 10 mL. Filter sterilize using 0.22 μm filter and store at 4℃. 

 - Beckman SW41 rotor

 - Centrifuge tube (Beckman Coulter, cat. 344059)
 

Detailed Procedure

1. Virus amplification
   -One day before infection, 2.5 million BHK-21 cells are inoculated in the T75 flask (8 T75 flasks in total) and cultured with 10% FBS DMEM;
   -The second day, the culture medium is replaced with 2% FBS DMEM and BHK-21 cells are Infected with VEEV-RABV-G virus at a MOI of 0.01;
   -Supernatant is collected at 96 hpi and placed in 50 mL centrifuge tubes when obvious CPE was observed;
2. Virus concentration and purification
   Collected supernatant was sequentially subjected to:
   -Centrifuge at 400 g for 10 min at 4℃;
   -Centrifuge at 5000 rpm for 20 min;
   -Filter the supernatant with 0.22 μm filter and place in a 50 mL Nalgene centrifuge ware (Thermo scientific, cat. C1295389).
   -The supernatant was added with 40% (W/V) PEG8000 to make the final concentration at 8%, and precipitated overnight at 4℃;
   -Centrifuge at 14,000 g for 1.5 h at 4℃, discard the supernatant and suck up the residual liquid;
   -Resuspend precipitation with 1 mL PBS in total, and the total volume after resuspension is about 1.3 mL;
   -Layered on top of 4.5 mL 10% sucrose cushion in centrifuge tube and centrifuged at 38,000 rpm for 1 h in a Beckman SW41 rotor. The pellet was resuspended in 100 μL of PBS.