Purification of Recombinant Virus Particles from Transfected Human Kidney Cells

Begin with a cell lysate containing cellular DNA aggregated with cellular proteins and viral particles.

Add magnesium ions, followed by a nuclease, to activate enzymatic degradation of cellular DNA and disrupt DNA–protein aggregates.

Centrifuge to separate debris and collect the supernatant containing viral particles.

Layer the supernatant onto a discontinuous iodixanol gradient with concentrations increasing from top to bottom, then fill the tube with buffer.

Ultracentrifuge to separate the components based on density. Virus particles accumulate at the interface between the high and medium-density layers.

Insert a needle at this interface and collect the viral particles into a tube.

Add a buffer containing stabilizing components, which prevents viral particle aggregation.

Then transfer the solution to a tube with a membrane filter.

Centrifuge to concentrate the viral particles above the filter.

Add the stabilizing agent-supplemented buffer and centrifuge again to obtain purified viral particles.

To harvest the transfected cells, dislodge and suspend the cells by using the culture medium to pipette up and down. Then transfer the cell suspensions to sterile 50-milliliter tubes. Centrifuge the