I've been using the baculovirus expression system for a year, but recent three months, I've seen the P1 and P2 virus grow white stuff inside. They are good within the first week and the protein expression is perfect. But after two weeks, I've seen some stuff floating in the virus solution and my protein yield drops abnormally. Has anyone else experienced this?
Hola, Lan. It seems that you have a fungus contamination in the parental viral DNA. or in plasmids or transfection agent. Seed a petri dish with rich medium with your passages to isolate and see the colonies, and filter by 0.22um all the component that you suspect could be contaminate.I hope this can help you. Good luck
Certainly sounds like a fungal contamination. I would throw out your viral stocks, your media, transfection reagent and re-prepare your bacmid DNA taking care with sterility.
One methods it might be worth looking into is storing your baculovirus as BIICs - baculovirus-infected insect cells. You basically harvest cells infected with your baculovirus just prior to lysis and freeze them down and use those to infect cultures for expression.
There is a publication here that details the methods (and many more methods online):
https://www.ncbi.nlm.nih.gov/pubmed/19174191
Much less chance of viral stocks going off and also deals with any issues of viral stability
Thanks everyone. A few weeks ago when this problem appears, I took out the white stuff and looked at it under the scope, it looks just like fibers. Also, I've filtered the virus with 0.22um again, but it appears the next day... But yesterday, I just tested the expression of protein, amazingly, the protein yield is similar as before again. Also, I contacted the media company about this problem, they said other customers and themselves also had seen this, which is not uncommon. So, luckily, the virus still can work.
Why HIV use reverse transcrition as it has positive sense ssRNA genome like group IV viruses?
It's really late to answer this hit but I have expreienced the same thing which made me start everything from the scratch. Nothing has changed. Athough I am a person who practice sterile techniques at high standard, I had seen some fiber-like things in my stocks and filtered them (0.22um) and next it appared again. So I decided to filter my stocks everytime before use them for infection. Yes virus is OK but if I did not filter it and start an infection the contamination will be getting worse and cells will just dye because of contamination not due to infection or as the result of CPE. hopefully it'll work fine.
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