Initial steps in the development of a new family of
allergy vaccines
Yin Jun
Allergic disease occurs when an individual react abnormally to allergens. Not only
human, but also animals can have allergic disease, for example, dogs, whose allergic
disease are very similar to human allergies. Unfortunately, most of medications for
allergies can only target allergic symptom, but not for curing. New treatment methods
should therefore aim at acting at a more early stage of the disease and also to try to
cure the disease. Therefore, we have initiated the development of a new class of
allergy vaccines.
Many existing researches have shown that the following three cytokines, namely
interleukin (IL)-18, IL-33, thymic stromal lymphopoietin (TSLP), play important
roles in allergic disease. They are the regulators of two white blood cell subsets (Th1,
Th2) balance, which has significant impacts on allergic diseases. The common
vaccine mechanism works like this, injection of the vaccine antigen into patient
stimulates white blood cells to divide and produce antibodies and the individual may
become resistant to the disease. However, since the three mentioned cytokines are
self proteins in our bodies and white blood cells could not react to them, a foreign
protein thioredoxin (Trx) is necessary to combine with cytokines in vaccines.
The aim of this study has been to optimize production, refolding and purification
protocols for the thioredoxin-dog IL-18 fusion protein. The second purpose of this
study was to perform the initial steps in the construction of bacterial expression
vectors for the production of thioredoxin IL-18, IL-33, TSLP fusion proteins for
several different species in E.coli.
During purification of dog-thioredoxin-IL-18 monomers, the following four steps
were done consecutively: denaturing, refolding, Ni+-NTA agarose affinity
chromatography and size exclusion gel chromatography. The first three steps were
successful but last step was failed unfortunately. All proteins were still aggregated
after purification, the reason could be that cysteine bridges in the monomers
interacted with thioredoxin amino acids.
In protein construction experiments, the following steps were done consecutively:
The individual fragments containing coding regions of dog, human and rat TSLP,
IL-33 and IL-18 were excised from the cloning vector and then were inserted into
the bacterial expression vector through ligation. Then the ligation samples were
taken up by competent bacteria to express expected proteins. The results showed that
coding regions of IL-33, IL-18 and TSLP were excised successfully and subcloning
was done.
In the future, new foreign proteins should be considered instead of thioredoxin to
avoid cysteine interaction problem, the IL-18, IL-33, TSLP cytokine vaccine could
continue to try to be expressed in the lab.