Identification Of Virulence-related Membrane Protein Complex From Shigella Flexneri 5a M90T

S.flexneri is a Gram-negative pathogen, which infects the host by virulence protein via all kinds of secretion system. So far, six kinds of secretion systems have been found. Though they are distinctive in secretion beaus and ways, they take shape in a germ membrane-across channel by single protein or macromolecules complex. As membrane complexes is complicated, strong hydrophobic and difficult to isolate integrally and purify from lipid bilayer of membrane, for a long time, only its structure and function of single membrane protein were studied on. In recent years, Shaegger have elected mature Blue Native Ployacrylamide Gel Electrophoresis(BN PAGE),which is breakthrough for complex research.S.flexneri acquiring the virulence plasmid is an important course to make it change from non-pathogenic into pathogenic. Virulence-related membrane protein complexes is very important for shigella's pathogenesis, such as Type III secretion system. Here is an efficient method to isolate membrane protein complexes from S.flexneri 5 wild type strain M90T and its large plasmid cured mutant M90T△T. A wild type specific band was found in complexes samples of M90T by Blue Native PAGE (BN PAGE) separation and comparison, which is about 290KDa and so named M90T-290. It was then dissociated into subunits by second dimensional SDS-PAGE and identified by MALDI-TOF MS. Six subunit proteins were identified, including a virulence large plasmid encoded protein (apyrase) and several chromosome encoded proteins. These proteins may form a novel membranC complex, which is involved in unipolar distribution of IcsA and intracellular spread of shigella.After the complex identification, we want to validate the composition of M90T-290 through affinity tag and purified technology. A recombination, which is composed of PGEX 6p-1 and phoN2, was constructed and transferred into the M90T, having been named M90T-6p-apy. The membrane complexes are fished by glutathione transferase. And then the complexes, which is eluted by glutathione of reduce, is isolated though BN-PAGE and SDS-PAGE. But no band is found in the experiment. Maybe GST tag altered the structure of apyrase and restrained the forming of the complex.In this study, A recombination, which is composed of PET 32a and phoN2, was constructed and transferred into the BL21,having been named BL-PET-apy. The supernatant of BL-PET-apy was found apyrase-his fusion protein.then purificated the supernatant by nickel pillar, which is filled with nickel sulphate.At the same time, in order to explore the function of apyrase and the complex we use the Red recombination system to knockout the gene phoN2, which coded the apyrase. Firstly, linear target DNA fragment was constructed and putted into the PKOBEG. We have been filtered the recombination bacteria, maybe the recombinant efficiency is too low.