Abstract:
Increased resistance to antibiotics and inefficient vaccines
prompted the development of new vaccines against the typhoid bacterium
Salmonella typhi. The object of the study was to clone and transform the
antigenic hypervariable regions IV to VI of the S. typhi flagellin gene in
the yeast expression vector pPICZaA, which will facilitate the production
of stable recombinant yeast cells for the formulation of new vaccines.
Plasmid pGTE-PCRII p23 containing the flagellin gene fragment and
pPICZaA were isolated from Escherichia coli strain JM-109 and then
subjected to restriction enzyme analysis using EcoRI/Xbal. The identified
flagellin gene fragment was ligated to the yeast expression vector
pPICZaA and the new construct was transformed into the E. coli strain
DH5a. The transformants were selected based on their resistance to the
antibiotic ZeocinTM and analyzed by restriction enzyme digestion with
BgnI, EcoRI/Xbal, and HindIII/EcoRI. The new constructs, designated as
pEO, were confirmed to be of the expected size - approximately equal to
4.1 kb. The pEO plasmids were isolated and transformed into the Pichia
pastoris strain X-33 through electroporation (5 milliseconds at 1.5 kV)
and the lithium chloride method. However, transformation in P. pastoris
was unsuccessful. This may be due to one or several factors, such as
technical and procedural errors.
