Choosing a Vector

The second component of the vaccine is the vector (plasmid backbone). The one major criterion here is that the vector must be strongly and constitutively expressed in mammals.

Ideally, the vector is specifically designed for protein expression in mammals: i.e., it has a leader (signal) peptide, a Kozak sequence, a transcription termination signal, and optimizations to improve expression, such as enhancers.

Many vectors include myc epitopes and/or polyhistidine tags to enable you to recover the recombinant protein. With a DNA vaccine, we are not attempting to harvest the recombinant protein so this stuff is unnecessary, and may even be detrimental.

I first looked around for a suitable BioBrick vector, but I could not find one. I found a reference to addgene.org that seemed promising, but the plasmids I found there were limited to non-commercial use.

Although I'd prefer not to use a commercial vector, there are a few that seemed promising.

• Invitrogen/Life Technologies pSecTag2
• clonTech pIRES: The main advantage of this vector is that it can express two proteins from one CMV promoter thanks to an IRES
• NEB pMCP

Protein Design

Like the Slovenian iGEM entry from 2008, I decided to use flagellin as my antigenic protein.

The Slovenian team were attempting to elicit a humoral response to H. pylori flagellin — a protein that normally evades the mammalian immune system — by creating a hybrid flagellin that is part E. coli and part H. pylori. This would then allow for the creation of a vaccine against H. pylori. At least half the world's population is infected with H. pylori, famously discovered as the cause of stomach ulcers.

Flagellin is about 400 amino acids long, which turns out to be helpful since it's still a little complex to synthesize anything longer than 2000 nucleotides. For example, IDT's gBlocks, which are very inexpensive and commonly used, max out at 2000 nucleotides. It's not particularly difficult to combine fragments to make longer genes, but it's also not off-the-shelf.

E. coli flagellin is highly immunogenic, as you might expect from a common and abundant bacterial protein. There's even a specific innate immune receptor, Toll-like receptor 5, that recognizes flagellins.

So as a short-ish protein that evokes a strong immune response, flagellin is a reasonable choice for my experiment.

LaTex

I started a LaTex document to describe my first DNA vaccine experiment in detail. It may also potentially be usable as part of an SBIR grant. My hope is that the LaTex document will be a more formal summary of information written up for this blog.