[The cloning and expression of the phospholipase D gene from Corynebacterium pseudotuberculosis]

Abstract

In a number of consecutive gene engineering operations a DNA fragment having a size of about 2.8 kb was cloned in Escherichia coli by means of Blue-script II SK+ used as vector. The insert contained pld gene coding the synthesis of phospholipase D, one of the key factors of C. pseudotuberculosis virulence. The stable and active expression of this gene in E.coli was achieved. High phospholipase A activity was accumulated in the periplasmatic space. The molecular weight of the synthesized protein was 31 kD. The product obtained by gene engineering methods was found to possess the biological activity of the natural product: it induced the hemolysis of sheep red blood cells in the presence of equi factor of Rhinococcus equi and inhibited the hemolytic activity of Staphylococcus aureus beta-hemolysin (phospholipase C). The pld gene cloned in these experiments differed from that of another C.pseudotuberculosis strain. Further research is underway with a view of searching for the limits of pls gene.