Construction Of Gene Knock-out Mutants Of Components Of Virulence Related Membrane Protein Complex Of S.Flexneri M90T

Shigella spp.are common bacterial pathogens causing human diarrheal diseases, seriously affecting people’s daily lives, severe cases, death. Of Shigella, pathogenic mechanism and host immune protection mechanism is not very clear, has not yet developed a very effective vaccine, can not effectively control the infection.Bacterial membrane protein complexes play an important role in the process of bacterial pathogenesis, The virulence plasmid is the key to the pathogenesis of Shigella, Shigella virulence plasmid cured mutant lose pathogenicity, wild strain and the virulence plasmid cured mutant membrane protein complexs are different, if differences, the differences between the membrane protein complexs involved in the pathogenic process of Shigella or Shigella virulence. In our laboratory as a starting point, Shigella flexneri5M90T wild strain and M90T virulence plasmid cured mutant membrane protein complexs spectrum were isolated by blue native PAGE (BN PAGE),and we found a unique band of wild strain in the molecular weight of about290kDa by comparing the observation, and this band were identified by MALDI-TOP MS, including six protein of a virulence plasmid encoded protein Apyrase and chromosome encoded protein DnaK、ClpB、GapA、 EF-Tu and YdgA.Apyrase and DnaK can affect the distribution of the polarity and function of the outer membrane protein IcsA, weakening the virulence of Shigella. Comprehensive analysis, we speculate that this complex proteins may jointly regulate the distribution of the polarity and function of the outer membrane protein IcsA.To verify our speculation, the present study, using Datsenko and Wanner have elected λRed recombination system, successfully knocked of apyrase, dnaK, c1pB, ydgA and icsA,has laid a good foundation for the next step. The same time, construction the process of gene knock-out mutants to explore optimization, master efficient, stable operation.