Abstract

Since protective immunity against shigellosis is serotype specific, it appears that construction of a live Shigella dysenteriae type 1 strain (attenuated by deletion of the virG gene as a native new vaccine candidate) is urgently needed. The virG gene from a native of S. dysenteriae type 1 was amplified and cloned into the pGEM-5Zf vector and sequenced. Alignment analysis indicated that the virG gene was 100 identical with other shigella serotypes. The polymerase chain reaction fragment carrying a chloramphenicol resistance cassette flanked by short (46 nt) regions homologous to the virG gene was electroporated into recipient S. dysenteriae strains expressing a highly proficient gamma beta exo gene system. This cassette allowed successful disruption of one invasive plasmid locus. The results obtained showed that 46-nt homology between the PCR product and the virG gene is enough to promote homologous recombination via the lambda Red system. The PCR and sequencing analysis demonstrated that 3220-nt of virG gene was successfully deleted. To intensify recombination efficiency, however, we used 232 ng of purified PCR-product. Upon Sereny test challenge with a native wild S. dysenteriae type 1, Delta virG-vaccinated animals were significantly protected against keratoconjunctivitis. The lambda Red recombinase is a new tool, powerful, inexpensive, rapid and simple method for the construction of in vivo genetic engineering.