The Study In The Relativity Of ArgT Protein To The Virulence Of Shigella Flexneri 2a

Shigella flexneri 2a has virulence at 37℃,and has no virulence at 30℃ because of the lose of its invasiveness. It had shown that Shigella flexneri 2a highly express ArgT protein at 30℃,yet this protein has a very low expression at 37℃.There have no relative reports about the expression at a lower temperature of ArgT protein, so it has both important significance and challenge to study the relationship between ArgT protein and the virulence of Shigella flexneri 2aObjectiveTo study the relationship between lysine-, arginine-, ornithine-binding periplasmic protein (ArgT) and the virulence of Shigella flexneri 2a .To explore the potential mechanism of how ArgT protein has a higher expression at a lower temperature and its influence on the growth of Shigella flexneri 2a.Methods1. The construction of the insertion mutant of Shigella flexneri 2a 2457T argT geneWe selected a sequence about 440bp on argT gene as a homologous sequence used by homologous reconstruction, that was argTI. The sequence argTI amplified by PCR was linked with pMD18-T clone vector, and then the recombinant plasmidpMDargTI was detected by ampicillin antibiotic, blue-white screen after it was transformed into the competent cells DH5a,then was digested by BamH I and Sal I .The cloned fragment argTI was cloned into suicide plasmid pXL275 which was also digested by the same two restriction endonucleases. The recombinant plasmid pXLargTI was detected by chloramphenicol and streptomycin antibiotic after it was transformed into the competent cells S17.1A.pir.245 7T wild strain and the recombinant were admixed at the ratio 1 I 1 on the LB plate which had no antibiotic. After cultured about 5h, the lawn was washed by fresh liquid LB and attenuated to 10 times, 100 times and 1000 times, then the liquid was spread on the chloramphenicol and nalidixic acid antibiotic LB plate and the positive integrant 2457TargT~ was screened by the two antibiotic.To verify the mutant 2457TargT~ by Southern blotting, we extracted the chromatosome of 2457T, and labeled argTI with digoxin as probe. Then the probe and the chromatosome of 2457T were hybridized.2. Observation the changes in the virulence of 245 7T by guinea pig keratoconjunctivitis experiment2457T wild strain cultured at 30 °C was dropped into one side of the first guinea pig' corneas, and the other side was the mutant 2457TargT~ cultured at 30°C. the bacteria cultured at 37°C were dropped into the second guinea pig' corneas, one side is 2457T wild strain, the other side is 2457TargT. The changes on corneas were observed after the bacteria were inoculated into the corneas.3. To observe the difference of the growth speed between 245 7T wild strain and the mutant 2457TargT~,we drew the growth curve of the bacteria cultured at both 30 °C and37°C.4. The comparison of the protein expression difference between 2457T wild strain and the mutant 2457TargT by two-dimensional electrophoresisWe prepared the whole cell protein samples of the two strains. First, the whole cell protein samples were separated by isoelectric focusing electrophoresis, then were separated by SDS-PAGE. After the electrophoresis was completed, the gels were scanned. We compared the gels image of the two strains, and searched those proteinswhich had different expression between the two strains, were cut by enzyme in gel, then These proteins were identified after the peptides mixture of every proteins were detected by MALDI-TOF mass spectrum and Mascot program. Results1. The insertion mutant of Shigella flexneri 2a 245 7T argT gene was successfully constructed.2. The change in the virulence was not so obvious to be observed after argT gene was inserted inactive at both 30°C and 37°C.3. The growth speed of the mutant was slower than the wild type strain at both 30°C and37°C.4. 19 proteins which had different expression were detected by 2-D electrophoresis,16 of these protein were identified by MALDI-TOF mass spectrum.Conclusion1. ArgT protein possibly had no direct relativity with the virulence of Shigella flexneri 2a 2457T.2. ArgT protein had important function on the growth of Shigella flexneri 2a 2457T.3. Those proteins that had different expression between 245 7T argT mutant and 2457T wild type strain were likely to have relationship to the function of ArgT protein.