Abstract
Helicobacter pylori (H. pylori) is a major pathogen involved in gastritis, peptic ulcer disease, and gastric cancer. Helicobacter pylori neutrophil-activating protein (HP-NAP) is an important virulence factor of H. pylori. The inflammation of the gastric mucosa caused by H. pylori infection might be resulted from the cytokines and reactive oxygen species (ROS) produced by HP-NAP-stimulated human leukocytes. Thus, H. pylori-induced inflammation of the gastric mucosa could be attenuated by blocking the activity of HP-NAP. Here, I found that antibody X not only detected their target protein but also detected recombinant HP-NAP. By western-blot, enzyme linked immunosorbent assay (ELISA) and native western-blot analyses, the antibody X detects denatured and native form recombinant HP-NAP of H. pylori 26695 strain. To determine the epitope sequence of the antibody X on HP-NAP, HP-NAP mutants were generated by using the modified PCR-based site-directed mutagenesis method and then purified by one-step DEAE anion-exchange chromatography. The antibody X is able to recognize HP-NAP through a new set of epitope sequence which is different from the original epitope of antibody X. The epitope sequence is conserved in all H. pylori strains. The non-identical amino acid residues which nearby the epitope sequence of HP-NAP in various H. pylori strains were then subjected to site-directed mutagenesis. I found that the antibody X could detect these mutated HP-NAP, indicating that antibody X is able to detect HP-NAP of various H. pylori strains. Furthermore, antibody X is able to inhibit HP-NAP-stimulated ROS production by human neutrophils. Thus, antibody X is able to detect HP-NAP and block its activity through the new epitope sequence of HP-NAP.