title: Helicobacter pylori recombinant UreG protein: cloning, expression, and assessment of its seroreactivity.
abstract: BACKGROUND
  Helicobacter pylori is a human pathogen and during the process of infection, antigens from the bacterium elicit strong host humoral immune responses. In our previous report, native H. pylori UreG protein showed good reactivity with sera from H. pylori patients. This study was aimed at producing the recombinant form of the protein (rUreG) and determining its seroreactivities.
  
  
  METHODS
  The coding sequence of H. pylori UreG was cloned and the recombinant protein expressed and purified by affinity chromatography using nickel nitrilotriacetic acid (Ni-NTA) resin. The antigenicity of rUreG to detect H. pylori specific antibodies was determined by western blot, using HRP-conjugated anti-human IgG and IgA antibodies as probes. A total of 70 sera, comprising 30 positive and 40 control serum samples, were used. The positive sera were from culture-positive H. pylori-infected patients with duodenal ulcers, gastric ulcers, or gastritis. The control sera comprised three types of samples without detectable H. pylori antibodies, i.e. healthy individuals (with no history of gastric disorders) (n = 10); patients who attended an endoscopy clinic (because of gastrointestinal complaints) but were H. pylori culture negative (n = 20); and people with other diseases (n = 10). Additionally, hyperimmune mice serum against rUreG was raised and tested with the native and recombinant UreG protein.
  
  
  RESULTS
  The ureG gene fragment was successfully cloned and expressed in both soluble and insoluble forms. Western blots on rUreG protein showed 70% (21/30) and 60% (18/30) reactivity with patients' sera when probed with HRP-conjugated anti-human IgG and IgA antibodies, respectively; and the combination of the IgG and IgA western blots showed reactivity of 83.3% (25/30). By comparison, 97.5% and 92.5% of the control sera showed no reactivity when probed with HRP-conjugated anti-human IgG and IgA antibodies, respectively. Both the H. pylori lysate antigen and rUreG protein displayed a distinctive band at the expected molecular weight when probed with the hyperimmune mice serum.
  
  
  CONCLUSIONS
  The rUreG protein was successfully cloned and expressed and showed good reactivity with H. pylori culture-positive patients' sera and no reactivity with most control sera. Thus, the diagnostic potential of this recombinant protein merits further investigation.
authors.forename: Akbar
authors.initials: A
authors.lastname: Khalilpour
authors.forename: Sabariah
authors.initials: S
authors.lastname: Osman
authors.forename: Muhammad Hafiznur
authors.initials: MH
authors.lastname: Yunus
authors.forename: Amutha
authors.initials: A
authors.lastname: Santhanam
authors.forename: Nagarajan
authors.initials: N
authors.lastname: Vellasamy
authors.affiliation: Institute for Research in Molecular Medicine (INFORMM), Universiti Sains Malaysia, 11800, Minden, Penang, Malaysia. rahmah8485@gmail.com.
authors.forename: Rahmah
authors.initials: R
authors.lastname: Noordin
mesh_terms: D000818:Animals; D000907:Antibodies, Bacterial / Q000097:blood; D000918:Antibody Specificity; D001426:Bacterial Proteins / Q000096:biosynthesis / Q000235:genetics* / Q000276:immunology* / Q000302:isolation & purification; D015153:Blotting, Western; D002352:Carrier Proteins / Q000096:biosynthesis / Q000235:genetics* / Q000276:immunology* / Q000302:isolation & purification; D016022:Case-Control Studies; D002846:Chromatography, Affinity; D003001:Cloning, Molecular; D000939:Epitopes; D016481:Helicobacter Infections / Q000097:blood / Q000175:diagnosis / Q000276:immunology* / Q000382:microbiology*; D016480:Helicobacter pylori / Q000235:genetics* / Q000276:immunology* / Q000378:metabolism; D006801:Humans; D007070:Immunoglobulin A / Q000097:blood; D007074:Immunoglobulin G / Q000097:blood; D051379:Mice; D008970:Molecular Weight; D028044:Phosphate-Binding Proteins; D011237:Predictive Value of Tests; D011994:Recombinant Proteins / Q000235:genetics / Q000276:immunology; D012698:Serologic Tests
chemical_list: D000907:Antibodies, Bacterial; D001426:Bacterial Proteins; D002352:Carrier Proteins; D000939:Epitopes; D007070:Immunoglobulin A; D007074:Immunoglobulin G; D028044:Phosphate-Binding Proteins; D011994:Recombinant Proteins; C082270:ureG protein, Bacteria