Hi, I am trying to make avian influenza virus VLP. I have a recombinant plasmid expressing HA,NA and M1 genes. I am using Drosophilla melanogaster S2 cells for these protein expression. When I do Western blots of the cell lysates I see HA and M1 protein bands. However,I do not see any bands in the supernatant. Is it normal to detect these proteins only on the cell lysates before sucrose gradient purification? Also, in order to see if these expressed HA,NA nad M1 proteins have self assembled to form VLP do I take the cell lysates or cell supernatant for the sucrose gradient purification? Also, what volume of culture do I need to grow the cells to in order to get decent amount of VLPs?
Your help and advices will be very much appreciated.
Dear Anita,
The band you see in Western blotting of the cell lysate are likely to result from expressed HA and M1 prior VLP formation. If you have VLPs they are supposed to be in the supernatant as well, as HA and M1 alone facilitate budding. So you should be able to detect them. If you do not see the proteins in Western blotting of the supernatant you do not have VLPs. As your S2 cell produce the proteins I guess something is wrong with the VLP budding.
Is it possible for you to use other cells like MDCK or HEK cells? They are normally used for Influenza VPL production and work quite well. And yes in order to purify VPLs you should use the supernatant for the sucrose gradient not the cells.
Good luck.
Thanks Jan.
There was one paper where they were using S2 cells to make HIV VLPs so I wanted to see if I am able to produce influenza VLPs using S2 insect cells, eventhough in 90% of paper people have used Sf9 insect cells and baculovirus system. Also, I am not cotransfecting three individual plasmids with HA,NA and M1 each, instead I have designed one plasmid containing HA,M1,NA separated by self cleavage peptides P2A and T2A:
-HA-P2A-M1-T2A-NA-
The self cleavage peptides are not 100% efficient in cleaving peptides. Do you think co transfecting individual plasmids is better than using self cleavage peptides?
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