Massive amplification of adenovirus in 293 cells

purified and identified, it can be amplified in large numbers in 293 cells. This manual provides a basic method for increasing the scale ofculture and the adenovirus infection cycle, according to which the amount of virus obtained is approximately 3×10¹¹ to 3×10¹² Since a cell contains 1000 to 10000 virus particles, 1L cultured cells can get about 5×10¹² virus particles. If the virus is to be centrifugalized with chlorpyrifod gradients, at least 3×10⁸ of cells must be used to correctly distinguish the virus belt.

For protein expression, it can be stopped at any step of amplification as needed, and protein pumping is carried out after centrifugal precipitation cells according to appropriate operating methods (depending on the type of protein, pick-up or cell precipitation). To optimize the time process, each infection cycle must be synchronized with the next cell amplification culture. If you cannot synchronize for a variety of reasons, you must freeze the virus until the next timecell culture begins and then melts the virus. Note that each amplification will leave some viruses that do not have to be discarded so that they can be spared if the next augment fails. Each amplification is best caused by the lowest generation of virus particles, which greatly reduces the likelihood ofmutated virus.

operating steps:

1 in 100mm Petri dish or 75cm^×2 < 10 ml DMEM5% culture 5×10⁶ 293 cells > "

2 takes 0.5ul's first amplified viral preservation fluid, adds DMEM 5% to 1 ml, mixes well, and dilutes the resulting MOI value of about 5.

3 Remove the culture fluid, carefully add the virus mixture, do not destroy the cell single layer, the cross slowly shake 3 times, 37 degrees C CO₂ incubator cultured for 90 minutes.

4 adds 9ml DMEM5%.

72 hours of culture, there are approximately 5×10^{< size>1" to 5}×10^{1" virus particles in a 1}0 ml solution for MOI determination to estimate virus particles×

: If the amount of virus obtained at this time is sufficient, virus titration can be performed immediately. Collect cells, 600×g centrifugation 5 minutes precipitate cells, add the smallest volume (generally 1/10 i.e. 1 ml of the original volume) virus to preserve the solution resusluated cells. -200C/370C freezes and melts 3 times, and the desktopcentrifuge centrifuges remove cell fragments at the maximum rate, collect them, and then titration the virus.

freezing and thawing 3 times from 6 -20 degrees C/37 degrees C.

7 into a 15 ml sterilecentrifuge tube and centrifuge at the maximum rate of 10 minutes on a desktop centrifuge, the collection is frozen at -20 degrees C or -80 degrees C.

10^{<1" cells were added to each of the 8 175cm2 culture bottles>7} 293 cells were cultured.

9 add 3 ml of cell lysate to 12 ml dmEM5% and mix well. Remove the cell culture, carefully add 5 ml of mixture to each bottle, shake the cross slowly and mix 3 times, 370C 5% CO2 incubator culture for 90 minutes. The MOI value is approximately 25 at this time.

10 to add DMEM 5% to 30ml.

48 to 72 hours at 11 a.m. At this time, the amount of 10 ml culture virus is about 3×10^{1" 10} to 3×10¹¹ . If necessary, MOI measurements are performed to estimate virus titration.

: If the amount of virus is sufficient for the experiment at this time, you can immediately enter the virus titration step. 600× g centrifugal 5 minutes to collect the cells, discard the clear, add 1/10 of the original volume of the solution rehanging cells. -200C /370C freezes and melts 3 times, and the desktop centrifuge precipitates cell fragments at the maximum rate, collects them, and titrifies the virus.

12. Move into a 50 ml centrifuge tube, centrifuge at maximum rate on the desktop centrifuge for 10 minutes, and remove the upper clear storage.

13. 30 bottles of 175 cm² each bottle is added to 10⁷ 293 cells.

14. Add 45 ml of cell lysate to 105 ml DMEM5% mix, remove the culture solution from the culture bottle, add 5 ml of mixed liquid to infect the cells, shake the cross slowly 3 times, 370C culture for 90 minutes. The MOI value is approximately 25 at this time.

15. Add DMEM 5% to 30 ml/bottle.

48-72 hours of culture, at which point there are approximately 3×10¹¹ to 3×10¹² viruses. If necessary, MOI assays are performed to estimate virus titration.

if you want to collect virus particles, first collect the infected cells and then resuscess in 5 ml DMEM5%. -200C/370C freezes and melts 3 times, centrifugal precipitation cell fragments. At this time 5ml DMEM5% of 3×10¹¹ to 3×10 ¹² viruses Particles, concentration of 6×10¹⁰ to 6×10¹¹vp/ml. The virus titration is then measured, or the virus is purified by the standard chlorpyrifode density gradient centrifugation method.

amplification of the virus in 10⁹ cells can obtain a large number of virus preservation fluids, while in 10¹ cell amplification is synthesic. Do not use the amplified viral preservation fluid again to infect cells, as this will greatly increase RCA production. Sometimes it is possible to use purified air spots to minimize RCA production. If there are no purified empty spots, then you have to re-empty spots purify the recombination virus, identify clones and then expand.

if you need to be larger than the amount of virus after 4 rounds of amplification, be careful to use early viruses as the source of amplification. For example, a second-generation virus is used to produce a third-generation virus. If there is no second-generation virus, a new second-generation virus must be produced with a first-generation virus. Amplification in accordance with this principle allows the RCA level to be as low as possible.

is used to express proteins, cells can be collected and appropriate methods can be selected to pick up proteins according to protein characteristics. Cell precipitation can generally extract 1-5 mg protein, it is recommended that after small-scale amplification to determine the infection after the optimal time to extract the protein, and then a large amount of amplification protein.

adenovirus amplification