Determination of Helicobacter pylori Immunogenes Using Two-Dimensional Gel Electrophoresis and Immunoblotting

Background and objective: Helicobacter pylori is one of the most common infectious agents that colonizes in the mucus layer of stomach. This bacterium has been identified to be the etiologic agent of chronic active gastritis, peptic ulceration and gastric cancer. The present study was aimed to identify H. pylori immunogenes for clinical diagnisis of the infection in the above ۳ groups of patients. Materials and Methods: H. pylori bacteria isolated from biopsy specimens of patients suffering from gastritis, peptic ulcer and gastric cancer were extracted in an extraction solution containing lysozyme, urea and CHAPS. Two-dimensional gel electrophoresis were performed. The resolved proteins were transferred to PVDF membrane using tank blotting and their reaction with purified IgG fraction of the patients’ sera were determined by immunoblotting. Results: The bacterial extract showed several hundreds of silver-stained spots with molecular weights (MW) ranging from ۱۰ to ۱۰۰ KDa and isoelectric points (pI) ranging from ۳.۵ to ۹.۵. This pattern contained ۶-۷ major proteins, some of which as protein groups consisted of several spots. The results of immunoblots revealed that several protein spots with different MW and pI, were stained with all three groups of patients’ sera but some proteins were stained only with one or two groups of sera. Conclusion: The protein spot with MW of ۳۰ KDa reacted with sera of only two groups of patients; gastritis and gastric cancer; the protein with MW of ۱۸ KDa reacted only with sera of gastritis patients. These proteins can be potential candidates for recognition of the type of gastric disorder. In addition, the results indicated that protein profiles of H. pylori, isolated from gastric cancer and peptic ulcer, are more similar to each other, comparing to that of gastritis patients.

Keywords:

Helicobacter pylori , Two-dimensional gel electrophoresis , Immunoblotting , Immunogens.