Alteration in apyrase enzyme attenuated virulence of XXXX .

@entity6835 @entity7913 @entity9849 @entity840 @entity969 @entity14341 @entity18815 @entity526 @entity2383
UNASSIGNED: Virulence of Shigella is attributed to the genes presence in chromosome or in the megaplasmid. The apy gene which is located in the megaplasmid of Shigella species encodes for apyrase enzyme, a pathogenesis-associated enzyme causing @entity2383 and host cell death. In this study we constructed an apy mutant of @entity14341 by insertional activation using a @entity7913 resistant gene cassette. The wild type apy gene of @entity18815 was PCR amplified, cloned and mutated with insertion of @entity7913 resistant gene cassette (aphA). The mutated construct (apy: aphA) was subcloned into a conjugative suicidal vector (pWM91) at the unique Sma1 and Sac1 sites. The mutation of the wild apy gene in the construct was confirmed by DNA sequencing. The mutated construct was introduced into wild type @entity18815 by conjugation with @entity526 . After undergoing homologous recombination, the wild apy gene was deleted from the construct using the @entity969 selection method. Non-functional activity of the apyrase enzyme in the constructed strain by colorimetric test indicated the successful mutation of the apyrase enzyme. This strain with mutated apy gene was evaluated for its protective efficacy using the @entity840 @entity9849 model. The strain was Sereny negative and it elicited a significant protection following challenge with wild @entity14341 . This apy mutant strain will form a base for the development of a vaccine target for @entity6835 .
