My feckin' vaccine

[LordZach 0]

destroyed DNA? sounds like it might sting a little bit.

[Hilandor 1]

i thought a marker virus was a method of being able to check to see if a subject has had the virus and had the innoculation against it because he says there is no way to find out if chcken had the innoculation or not then a marker would show up when chicken was tested then they would know if it was innoculated or not.

ahh i got nightmares at the mention of elisa test and western blot i did a bit of that at college and it was wayy over my head.

[Gorgi Knootewoot 0]

</span><table border="0" align="center" width="95%" cellpadding="3" cellspacing="1"><tr><td>Quote (supah @ May 17 2002,11:56)</td></tr><tr><td id="QUOTE">Gorgi, just curious. What exactly is a marker vaccine? does it just show that the chicken has the virus? does it make its feathers turn blue or something? or does it also kill the virusses? just wondering.<span id='postcolor'>

A marker-vaccin is that you have changed something in the thing u used for the vaccine..

A vaccin can also be toxines, bacteria, little woms in your body where i don't know the word from, and of course virusses.

I changed a protein in the virus, so that i can distinguish this altered virus form the field-strain (the unaltered virus)

When using this altered virus as a vaccin, and you have a test to distinguish it from the other virus, it is called a marker-vaccin. The vaccinated chickens will look the same as the ill ones. Therefore a bloodsample must me takes from each chicken, and tested using the method that will tell the difference between the both virusses.

[Gorgi Knootewoot 0]

And there are lots of different marker vaccines. For the pigs disease Aujesky, all infected pigs have antibody's against the viral protein gI. The marker-vaccin misses this protein (they removed it), so when a pig is vaccinated and they want to tell the difference between an infected pig and a vaccinated one they do a small bloodtest to see if there are antibody's against this gI. If not, the pig is healhy and vaccinated.

[Oligo 1]

Oh, so it's a marker vaccine that you made. Nice one. Since you talked about changing RNA, I assume it is a retrovirus, or?

It's kind of interesting, since I also work with viruses, although for completely different purposes. I'd like to know (if you can tell) whether this virus you altered has a big genome (over 10000 bases) and if so, how did you alter the genome? Did you use recombination or restriction digestion (cut out the gene to knock it out)? Maybe you just knocked out the gene with point mutagenesis? Or something else? It would be interesting to know, because I have noted that changing viral genomes can sometimes be quite hard.

[Gorgi Knootewoot 0]

</span><table border="0" align="center" width="95%" cellpadding="3" cellspacing="1"><tr><td>Quote (Oligo @ May 17 2002,13:01)</td></tr><tr><td id="QUOTE">Oh, so it's a marker vaccine that you made. Nice one. Since you talked about changing RNA, I assume it is a retrovirus, or?

It's kind of interesting, since I also work with viruses, although for completely different purposes. I'd like to know (if you can tell) whether this virus you altered has a big genome (over 10000 bases) and if so, how did you alter the genome? Did you use recombination or restriction digestion (cut out the gene to knock it out)? Maybe you just knocked out the gene with point mutagenesis? Or something else? It would be interesting to know, because I have noted that changing viral genomes can sometimes be quite hard.<span id='postcolor'>

I always thought that retro-viruses where the ones that produces DNA using reverse-transcriptase, and then make RNA. I could be wrong, but i have a RNA-virus but it isn't a retrovirus. (HIV is a retrovirus, and it makes first DNA, then RNA). But i also don't know which it is then.

It has a genome of only about 3100 bases. I did it the simple way of mutating the specific gene. It took a while, but there was a monoclonal antibody directed only to this protein. So i used a shitload of viruses and incubated them with this antibody. Almost every virus was down thanks to the antibody, but after a while some got a point mutation is their genome. They altered themselves. I watched them during a few month now, and most of them have fallen back to their original genome, and they are dead. I have 6 of the about 1000 left that are stable now. I'm trying to identifie the mutation soon by sequencing the genome. The part i want is only 400 bases, so it could be done in one time. (when using the methode of Sanger, and you have more than 600 bases it can go wrong when using only two primers.) I have to figure out which primers i should use, but that's not a problem. But time is running out (one more month) and i have to stabalize the virus for another 10 passages.

[Oligo 1]

I thought all RNA viruses are supposed to reverse-transcribe their genome to DNA once they have entered the host, since the molecular apparatus of animal cells cannot replicate RNA. Then again, maybe the virus has it's own proteins for the replication of the virus genome. I'm really not the expert on RNA viruses. Anyway, I do know that RNA replication is very very error-prone. The mutation rate of the virus you modified must be phenomenal.

From what you describe, I'd say that the molecular evolution you performed did not knock out the surface protein gene, but rather changed the amino acid sequence encoded subtly enough to prevent the antibody from binding, but retaining surface protein function. So you're probably going to find one or a few point mutation(s), which changes the encoded amino acid sequence subtly.

The molecular evolution approach you used (incubation with antibody to knock out unwanted types of viruses) sounds very elegant. I've used similar techniques with phage display to select for proteins with certain characteristics.

[FallenPaladin 0]

You can really learn something in this forum!

Many won`t believe that!

[Gorgi Knootewoot 0]

Yes you are right, the protein is still there. It has however a region that is a bit different from the original one, so that the monoclonal antibody doesn't bind anymore.

For detection i tried to use the Western Blot, but the monoclonal didn't bind to the original protein. I could only guess that the protein changed when it was run through the SDS-PAGE.

Now i use ELISA, and it works great. Simple methode like blotting, done in one day. (except i block overnight with skimmilk to block of any a-specific binding sites)

What methods did u use for the phage's ?

I think a plaque assay can be done with this...

[Oligo 1]

I used DELFIA, which is an immunoassay like ELISA, except the label is different. The principle, however, is all the same. I could nicely determine how the affinity between the antibody and the target protein had changed, because immunoassays allow for numerical affinity determinations (Scatchard plots). So I could nicely determine, whether the antibody bound the mutated protein with ten times or hundred or thousand times better or poorer than the native protein. Robust technique.