| Objective To explore a large-scale culture method for H.pylori and purify its urease. Methods Columbia Agar base (CAB) as plating media was used, in the process of recovery culture and subculture, to observe the growth of H.pylori when different nutrient supplements (10%SB, 10%SBS and 0.1% β -CD) were added, or when plates were exposed in the atmospere for different periods of time (1h and 3h), or when the different negative pressures (-0.075Mpa, -0.070Mpa and -0.065Mpa) were attained while pumping out the excess oxygen from the jar. Brain Heart Infusion broth (BHIB) was used as the liquid media to observe the growth of H.pylori when different nutrient supplements (5%CBS, 10%CBS, 10%SBS and 0.1% P -CD) were added. Calculations and comparison of the cost and the time consumed for 1g of H.pylori in different cuture medias were performed. Urease was extracted from the bacterium using a spin-surge and alternate freezing and thawing mothod and purified by a three-step chromatography of Q Sepharose FF anion-exchange, Phenyl Sepharose HP Hydrophobic interaction and Source 30S Sepharose cation-exchange. The enzymatic activity of the purified urease was detected with a spectrophotometic assay based on phenol red. The overall protein recovery was calculated. Identification of purified urease was performed by SDS-PAGE and Western blotting. Results From solid plate medias added with 10%SB, 10%SBS and 0.1% P -CD, 0.0618g, 0.0576g and 0.0335g of H.pylori was attained respectively after 4 days' growth of recovery and 0.0802g 0.0922g and 0.0447g of H.pylori was attained respectively after 3 days' growth of subculture. Miether it was during the recovery or the subculture, there's no statistic significance (P > 0.05) of H.pylori production between the 10%SB group and the 10% SBS group, but both were superior to the 0.1% P -CD group (P < 0.01); neither the different lengths of the exposed periods of plates to the atmosphere nor the different negative pressures when pumping out the excess oxygen from the jar revealed statistic significances (P > 0.05) in H.pylori production. The OD650 values of 0.2532, 0.3705, 0.3173 and 0.2336 were attained from the liquid medias added with 5%CBS, 10%CBS, 10%SBS and 0.1% P -CD respectively after 3 days' growth. Both the 10% CBS group and the 10% SBS group were superior to the 0.1% P-CD groupCP < 0.01), the 10% CBS group was superior to the 5%CBS group (P < 0.01). For lg of H.pylori, the expense and the relative times needed for the culture of the solid media added with 10%SBS was $24.23 and 1.00. The 10%SB group ($26.28 and 1.14) was smilar to the 10%SBS group. The final purified urease still had enzymatic activity of 19.7 mM urea/mg/min. The overall protein recovery was 2.58%. The final urease showed the typical subunits of M 66 000 (urease B) and M, 30 000 (urease A) when subjected to SDS-PAGE, and was recognized by rabbit anti-whole-cell of H.pylori serum. Conclusions Solid medias supplemented with 10% SB or 10%SBS can yield a relatively high H.pylori production. Large-scale and rapid urease purification can be performed by the three-step purification method. |
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