Inspiration
First, we noticed that dengue fever is severe disease which could affect almost half the world. We learned through literature and reports that dengue hemorrhagic fever and dengue shock syndrome have high mortality rates, and these result from the interaction between CLEC5A on macrophages and the envelope proteins (E proteins) of dengue virions.
Imagine if there were so enough proteins or antibodies to “disturb” the CLEC5A – dengue interaction, just like “smoke grenades” in our body? That would be a fantastic treatment option. It would take a long time to achieve, though.
Next, we thought about using peptides as “peptides cloud” to detect virions outside the body, since they are small and can be produced much faster than antibodies.
Design
We designed two parts, which we called “tandem repeat sequence-1” (TRS-1) and TRS-2, using the binding site of CLEC5A as a reference. We use template-repeat PCR (TR-PCR) as method of producing these peptides, which takes primers as PCR templates and produces a long chain of repeating sequences of peptide DNA. There is a problem that the primer concentration is highly sensitive to the peptide chain length (repeat times). We found the lower the concentration the longer the chain that will be produced.
In the next stage of the experiment, we use pET-29(b) as a vector of TRS-1 and TRS-2 to transform E.coli DH5α to clone more peptide sequences. After that, we then transform the chosen peptide sequences into E.coli BL21 (DE3) to express peptides.E.coli DH5α has an property of high transformation efficiency, and E.coli BL21 (DE3) can express protein or peptide effectively. So we choose these two different strains to do different parts.
In the initial design, we planned to add an arginine sequence to the end of the peptide sequence, which could be cleaved by trypsin at the arginine C-terminal of the peptide. Unfortunately, based on a calculation of the electrostatic force of gold nanoparticles, we decided we should use a longer peptide instead of the peptide monomer because there is an electrostatic force between the gold nanoparticles, so if one gold nanoparticle with TRS interacts with an E protein on the virion, the other gold nanoparticles might find it difficult to interact with the E protein. Thus, we designed the TR-PCR primer without an arginine sequence to produce the peptide and chose the size that is most likely to interact with dengue E proteins, according to Rosetta simulations.
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