| Purification of VacA toxin of Helicobacterpylori and the machanism of vacuolating toxicity in vitro(abstract)Helicobacter pylori(H.pylori) plays an important role in the pathogenesis of peptic ulcer, gastritis, MALT lymphoma. VacA (vacuolating cytotoxin) is one toxin secreted by the organism. The machanism of vacuolating toxicity is still unknown. Our aim is to search one simple , reliable and feasible method to purify VacA from H.pylori supernatants, through the comparison of liquid culture methods and column chromatography. The value of neutral red uptake(NRU) assay in detection of vacuolating activity was also evaluated. Above that we observed the effect of inhibitors of protein-tyrosine phosphatase(PTP) and protein-tyrosine kinase(PTK) on the vacuolating activity. The results could be used in the estimation of the role of VacA in the signal tranduction of cell, and the character of VacA receptor could also be proved.1. Rough extraction of VacA from the supernatants of H.pylori:Tryptic soy broth (TSB ) , Mueller-Hinton broth (MHB ), and two kinds of support reagents fetal bovine serum(FBS) and P -Cyclodextrin (CD) were used in our research. According to the component of Brucella broth, liquid culture of H.pylori was classified into four groups: TSB+FBS, TSB+CD, MHB+FBS andMHB+CD. H.pylori CCUG17874 was cultured in the above broth in an ambient atmosphere containing 5% O2 for 72h at 37癈. The culture results showed that density of H.pylori in TSB+CD broth reached 10.33?.76 * 108CFU/ml, which was lower than that of TSB+FBS ( 13.17?.15, P>0.05 ) . The concentration of protein in the extraction of supernatants of these two groups was significantly higher than that of culture containing MHB ( P<0.01 ). Result of SDS-PAGE showed the line of 95kDa(VacA) could be seen in the mixtureof groups containing TSB, but protein composition was more complex in groups containing FBS, especially the protein of 66kDa. VacA could not be seen in the extract of MHB supernatants.SGC-7901 cells were cultured in DPMI 1640 containing 10% FBS in 96-well plates. Toxin preparations were serially diluted in 1640, and were incubated with adherent cells and 1640 for 24h at 371C. The vacuoles in cells could be seen around the nuclear. Percent of vacuolated cells reached 100 % at 1:5 dilution, and decreased with the concentration. Cell vacuolation was then quantitated spectrophotometrically using a neutral red uptake(NRU) assay at 550nm. Dilution higher than 1:20 in group TSB+CD and 1:40 in group TSB+FBS could cause strong vacuolating toxicity. A550 of group TSB+FBS was 0.68?.07, which was higher than of group TSB+CD ( 0.54?.09) ,but the difference was not significant (P>0.05 ) . Vacuolating activity also changed with time. The vacuoles could be seen after 4h at dilution 1:2 (A550 = 0.43?.06 ) , and reached the maximum at 16h ( A550 = 0.71?.03 ) . A550 abnormally decreased at 24h due to the death of cells. The curve of dilution 1:5 reflected the character of vacuolation more correctly than of dilution 1:2. Results of NRU were in accord with the amount of vacuolated cells, and in some cases NRU had a higher sensitivity. 2. Purification of VacA:The purification of the vacuolating toxin of H.pylori involved ammonium sulfate precipitation of proteins present in broth culture supernatant, followed by hydroxyapatite, hydrophobic interactive, anion exchange and gel filtration chromatography. The 5ml dialyzed sample from one liter culture was applied to a hydroxyapatite column. After washing with lOmmol/L phophate buffer at a flow rate of 0.35 ml/min,-7-VacA was eluted in the first peak the max absorbance of which was 278mAU. Protein in the sample was then precipitated with a 50% saturated solution of ammonium sulfate, and dialyzed in 60mmol/L Tris( pH7.7 ) buffer. Hydrophobic interactive chromatography was performed on a phenyl-Sepharose CL-4B column with the same buffer containing 0.5 mol/L (NH^SC^ , and proteins containing VacA were eluted with 60mmol/L Tris. Anion exchange chromatography was performed...
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