Cloning and Transformation of Salmonella typhi Flagellin Gene in the Yeast Expression Vector pPICZaA

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.