Helicobacter pylori recombinant UreG protein: cloning, expression, and assessment of its seroreactivity

BMC Research Notes

Rahmah Noordin²  Nagarajan Vellasamy¹  Amutha Santhanam²  Muhammad Hafiznur Yunus²  Sabariah Osman²  Akbar Khalilpour² 

[1] Hospital Seberang Jaya, Penang, Malaysia;Institute for Research in Molecular Medicine (INFORMM), Universiti Sains Malaysia, 11800, Minden, Penang, Malaysia

关键词: Seroreactivity;    Recombinant protein;    Expression;    Cloning;    UreG;    Helicobacter pylori;   

Others  :  1125607
DOI  :  10.1186/1756-0500-7-809

 received in 2013-10-31, accepted in 2014-10-31,  发布年份 2014

【 摘 要 】

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.

Conclusion

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.

【 授权许可】

   
2014 Khalilpour et al.; licensee BioMed Central Ltd.

【 图 表 】

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