شناسايي و تخليص شاخص هاي آنتي ژني هليكوباكترپيلوري

Identification and purification of Helicobacter pylori antigenic determinant

Helicobacter pylori are the most important cause of chronic stomach infection, chronic gastritis, peptic ulcers and gastric cancer in men. This gram-negative microorganism is S-shaped, microaerophillic and has potent urease activity. After H. pylori ingestion, the microorganism attaches to the epithelial surface of stomach and is clonized. Subsequently, the physiologic and cytological damage is occurred. In the present work stomach biopsies were prepared by an experienced endoscopist physician, inoculated in transport media and sent to laboratory. Concurrently, 5ml blood sample was obtained from patients. Biopsy sample was homogenized in an aerobic sterile condition and then was cultured on selective media for 5to 7 days at 37 °C. the pure colonies were harvested from media and preserved in PBS sterile solution, then, samples subjected to ultrasound waves that caused bacterial lysis.12.5% SDSPAGE using wide range molecular weight marker was done. Lysed bacteria were divided to pure protein fractions by Gel filtration chromatography. Samples were quantified by spectrophotometer. Samples with high absorbance were incubated with positive sera and result was analyzed in regard to positive response in dot-blotting method. Antigenic profile of lysed bacterial electrophoresis indicates protein precipitation in 21,8 kilo Dalton area. These results indicate the ability of antigens in stimulating immune response. Also in negative samples, a lower antibody response was seen. Therefore intact bacteria are not suitable for serological diagnosis and only specific proteins should be used for this purpose.