Production of recombinant urease for screening of helicobacter pylori infection

Abstract

Helicobacter pylori establishes infection inside human stomach lining and causing duodenal diseases, such as peptic ulcer and potentially into gastric cancer. The invasive diagnostic methods require unpleasant endoscopic procedure for H. pylori detection. Preferably, the noninvasive diagnostic methods would make suspected patients less stressful to procedure for H. pylori detection. The two most preferable noninvasive diagnostic methods are antibody detection (serology) and antigen detection (from fecal) with their own advantages and drawbacks. H. pylori urease is one of the antigens found in H. pylori with strong immunogenic property. Thus, urease was chosen for the development of H. pylori dot-EIA test strip which could be used in a serology based detection system for H. pylori infection. Cloning of urease A gene fragment (pET32ureA3), urease B gene fragment (pET32ureB2) and the whole of urease operon (pET32UOA6) produced biologically active recombinant urease A (UreA), recombinant urease B (UreB) and recombinant urease enzyme complex (UreA/UreB) verified by immune functioning assay using commercial antibody H. pylori urease-α and commercial antibody H. pylori urease-β from Santa Cruz, Inc, USA. The production of recombinant UreA/UreB complex indicates that a fully functional urease operon or a urease replicon was successfully constructed. Purifications of the recombinant ureases were successful and the purified recombinant ureases were still biologically active. The purified recombinant ureases were coated onto membranes in preparation of dot-EIA test strips. The prepared dot- EIA test strips gave brown colour dots indicating positive reactions when probed with commercial antibodies against ureases (Santa Cruz, Inc, USA). Nine rabbits were used to assess the immunogenicity properties of the recombinant ureases. Sera from the challenged animals gave positive detections on the prepared test strips, similar to detections using commercial antibodies, indicating the recombinant ureases could act as immunogens comparable to native ureases. Three groups containing three rabbits each were challenged with purified recombinant UreA, UreB or UreA/UreB respectively showed the presence of ureases antibodies in their sera on dot-EIA test strips. One rabbit that served as a negative control, challenged with bovine serum albumin (BSA), did not give positive reaction on dot-EIA test strip. To conclude, this study was successfully achieving all the objectives: cloning, expression and purification of functioning recombinant ureases, as well as, developing dot-EIA test strip. With the intention to develop user friendly, easy, cheap and fast detection; this dot-EIA test strip provides a foundation for further urease enzyme-linked serology based assay development as a mean for early screening. In addition, the constructed H. pylori urease replicon opened an opportunity for developing a genetically modified animal model to study H. pylori pathogenesis.